Lightner Witmer was born in Philadelphia, Pennsylvania, on June 28, 1867. Lightner was the first of four children born to David and Katherine Witmer. David Lightner worked as a successful merchant, and the family lived comfortably.

His parents placed high esteem on education for their children. As a young boy, Lightner attended dance school to learn proper social etiquette. In September 1880, Witmer enrolled in the Episcopal Academy of Philadelphia, which was one of the top prep schools in America at the time. A common anecdote from this stage in Witmer's life demonstrates his lifelong lust for knowledge and improvement. Witmer and two other boys were instructed to build a canoe. Each boy had the proper instructions and materials. As the two other boys argued about who would finish first, Witmer thought, "I wish to finish last as I will learn from the others' mistakes and build the best canoe." Witmer graduated from the Academy on June 30, 1884.

In fall 1884, Witmer enrolled in the University of Pennsylvania (Penn). Witmer's freshman class was comprised of 24 young men comprised Witmer's freshman, and he was elected President. In 1888, Witmer graduated from Penn, receiving his A.B. degree.

In fall 1888, Witmer accepted a teaching position at Rugby Academy, a secondary school for boys. He taught English and History for two years. In his most famous article, "Clinical Psychology," Witmer reported about his experiences with a boy in one of his English classes at Rugby. The boy had language difficulties stemming from his trouble to distinguish between sounds. Witmer called the boy's condition "verbal deafness," which might be considered dyslexia today. With Witmer's one-on-one help, the boy improved his skills so extensively that he was able to enroll at Penn the next year.

While still teaching in fall 1889, Witmer entered Penn's graduate school in the philsophy department with the intention on working towards an advanced degree in political science. The same year that Witmer enrolled in graduate school, a new professor, James McKeen Cattell, joined the philsophy department's faculty. At the time, psychology was a newly-developing field. Cattell studied with both Wundt and Galton and was considered to be the world's best-trained psychologist. Witmer soon became Cattell's assistant. Together, the pair set up a psychology laboratory. Witmer's first experience in the lab required him to collect data detailing individual differences in reaction time. In fall 1890, Cattell left Penn for a higher paying position at Columbia University. When Cattell left, Witmer decided to go to the University of Leipzig.

Witmer planned to earn a Ph.D. under Wilhelm Wundt and to return to direct the psychology lab at Penn. He boarded The Russia destined for Germany in February 1891. After arriving in Germany, Witmer entered the philosophy program at Leipzig. While there was no designated psychology department, Wundt was the head of the philosophy program. Witmer investigated the aesthetic value of differing visual forms at Leipzig. Witmer used 14 objects and asked participants to assess which shapes were most appealing. Witmer's dissertation, "On the Experimental Aesthetics of Simple Spatial Relationships of Form," was published in Wundt's journal Philosophische Studien. Witmer also wrote an extensive abstract detailing his research in English. The abstract was printed in the Psychological Review, an American journal. Witmer graduated magna cum laude and was awarded his doctoral diploma on March 29, 1893. He also received the title of "Magister" for his mastery of psychology.

In fall 1892, Witmer returned to Penn as a lecturer in experimental psychology. He lectured and conducted laboratory work. Around this time, Witmer also decided to become a charter member of a new association for psychology professionals. In December 1892, Witmer presented two papers at the American Psychological Association's first meeting. Although the original copies of these papers have not been found, available abstracts hint at the papers' content. The first paper elaborated on studies following up Witmer's dissertation experiment while the second paper detailed his research on individual differences in reaction-time.

Witmer's professional interests began to shift from pure science to a combination of science and application. In 1896, Witmer founded the world's first psychological clinic at Penn. At the same time, Witmer began publishing papers such as "Practical Work in Psychology" that detailed the importance of applying scientific research to clinical work in the real world. When the United States declared war on Spain, Witmer enlisted voluntarily. He served in the Philadelphia City Calvary and was stationed in Puerto Rico. He was discharged the same year. Witmer's research interests turned towards "teaching to weakness." His work mostly focused on children's learning difficulties and how to improve their skills. His particular topics of interest were intelligence, speech problems, stammering, over-excitability, and nervousness among others. While still teaching at Penn, he also taught at Lehigh and Bryn Mawr.

In 1904, Witmer married Emma "Fifi" Repplier. Emma was a writer. Her work in literary research for the American Philosophical Society led her to meet Lightner.

Witmer continued his work with the clinic and advancing knowledge in academia. During the 1920s, the clinic saw almost 600 cases each month. With a $10,000 grant from a wealthy benefactor, Witmer's published the first issue of his journal—The Psychological Clinic. This journal is regarded as the first scholarly psychology journal. Witmer's article "Clinical Psychology" introduced this new term in the journal's first issue. The article described his last ten years of work. It also explained why the term "clinical psychologist" was needed and why psychology must include both research and practice. He also moved his workings beyond the realm of academia. From 1906 to 1956, Witmer held the title of "psychologist" for the Pennsylvania Training School for Feeble-Minded Children and also worked at the Devereux School—a boarding school for emotionally, developmentally, or cognitively impaired or delayed children. In order to further his research, Witmer developed a residential school in Wallingford, Pennsylvania, and later in Devon, Pennsylvania.

The last issue of The Psychological Clinic was published in 1935 when Witmer's professional career was winding down significantly. Witmer officially retired from Penn in 1937. Around this time Witmer and his wife separated, but they maintained an amicable relationship. Even during his retirement, Witmer maintained his involvement with the Devereux school in Devon. Lightner Witmer sufferred from heart failure and died on July 19, 1956, in a hospital in Bryn Mawr, Pennsylvania.