Eakins lived in Philadelphia. He graduated from Central High School (Philadelphia), studied at the Pennsylvvania Academy of Fine Arts, and then in Europe 1866-1870, notably with Jean-Léon Gérôme in Paris, though he spent time in Spain as well, enamored with artists such as Diego Velazquez. He returned to the Pennsylvania Academy to teach in 1876, later becoming the director of the Academy in 1882. His teaching methods were controversial, most notably his interest in instructing his students in all aspects of the human figure, including the nude. Though there were tensions between him and the Academy's board of directors throughout his teaching career, he was ultimately fired in 1886 for removing the lioncloth of a male model in a class where female students were present. The majority of Eakins's students liked his teaching methods and encouraged him to continuing teaching them at Philadelphia's Art Students League.

Eakins's early works upon his return from Europe include a large group of rowing scenes, and Eakins was a rower himself. The most famous of these is "The Champion Single Sculling" also known as "Max Schmitt in a Single Scull" (1871), now located at the Metropolitan Museum of Art. These early works involved critical observation of the painting's location, as well as preparatory drawings of the figure and perspectival plans of the scull in the water. Eakins painted himself into the Max Schmitt painting and can be found just to the right of the champion rower with his name inscribed on his own boat.

In the late 1870s Eakins was introduced to the photographic motion studies of Eadweard Muybridge, and became enamored with the possibilities of the camera. He performed his own motion studies, usually invovling the nude figure, and even developed his own technique for capturing motion on film. In 1881 he got his own camera and works after this point areknown to have photographic sources, such as "Mending the Net" (1881) and "Arcadia"(1883).

Some of his most well-known works include "The Gross Clinic" (1875) and "The Agnew Clinic" (1889) (both portraits of surgeons), as well as "The Swimming Hole" (1884-1885, also referred to as "Swimming").

Deeply affected by his dismissal from the Academy, Eakins's later career focuses more on portraiture than genre paintings. Eakins approached these portraits not with a scientific eye, but one that looks into the psychological presence of the sitter. Often his portraits were rejected by the sitter, as he often portrayed them years older than they really were. The majority of these portraits were of friends and family, very few of them were actually commissioned. His portrait of Walt Whitman was the poet's favorite.

Eakins's attitude towards realism in painting, and his desire to explore the heart of American life strongly influenced the Ashcan School. He was a teacher of early African-American painter Henry Ossawa Tanner. Though his is not a household name, and in spite of the fact that during his lifetime Eakins struggled to make a living from his work, today he is regarded as one of the most important American artists of any period.

Furthermore, since the 1990s, Eakins has emerged as a major figure in sexuality studies in art history, for both the homoeroticism of his work and for the complexity of his attitudes towards women. Controversy shaped much of his career as a teacher and as an artist. He insisted on teaching men and women "the same", used nude models in mixed sex classes, and was accused of abusing female students. Recent scholarship suggests that these controversies were grounded in more than the "puritanical prudery" of his colleagues (as has been assumed). Today, scholars see these controversies as caused by a combination of factors such as the boheminanism of Eakins and his circle (in which students, for example, sometimes modeled in the nude for each other), and Eakins's own personality (he was known to make rude jokes etc. to provoke people).