Eakins was born and lived most of his life in Philadelphia. He was the first child of Caroline Cowperthwait Eakins, a woman of English and Dutch descent, and Benjamin Eakins, a writing master and calligraphy Teacher of Scots-Irish ancestry. Benjamin Eakins grew up on a farm in Valley Forge, Pennsylvania, the son of a weaver. He was successful in his chosen profession, and moved to Philadelphia in the early 1840s to raise his family. Thomas Eakins observed his father at work and by twelve demonstrated skill in precise line drawing, perspective, and the use of a grid to lay out a careful design, skills he later applied to his art.

Eakins then studied art in Europe from 1866 to 1870, notably in Paris with Jean-Léon Gérôme, being only the second American pupil of the French realist Painter, famous as a master of Orientalism. He also attended the atelier of Léon Bonnat, a realist Painter who emphasized anatomical preciseness, a method adapted by Eakins. While studying at the École des Beaux-Arts, he seems to have taken scant interest in the new Impressionist movement, nor was he impressed by what he perceived as the classical pretensions of the French Academy. A letter home to his father in 1868 made his aesthetic clear:

A trip to Spain for six months confirmed his admiration for the realism of artists such as Diego Velázquez and Jusepe de Ribera. In Seville in 1869 he painted Carmelita Requeña, a portrait of a seven-year-old gypsy Dancer more freely and colorfully painted than his Paris studies. That same year he attempted his first large oil painting, A Street Scene in Seville, wherein he first dealt with the complications of a scene observed outside the studio. Although he failed to matriculate in a formal degree program and had showed no works in the European salons, Eakins succeeded in absorbing the techniques and methods of French and Spanish masters, and he began to formulate his artistic vision which he demonstrated in his first major painting upon his return to America. "I shall seek to achieve my broad effect from the very beginning", he declared.

Eakins' lifelong interest in the figure, nude or nearly so, took several thematic forms. The rowing paintings of the early 1870s constitute the first series of figure studies. In Eakins' largest picture on the subject, The Biglin Brothers Turning the Stake (1873), the muscular dynamism of the body is given its fullest treatment.

At the same time that he made these initial ventures into outdoor themes, Eakins produced a series of domestic Victorian interiors, often with his father, his sisters or friends as the subjects. Home Scene (1871), Elizabeth at the Piano (1875), The Chess Players (1876), and Elizabeth Crowell and her Dog (1874), each dark in tonality, focus on the unsentimental characterization of individuals adopting natural attitudes in their homes.

It was in this vein that in 1872 he painted his first large scale portrait, Kathrin, in which the subject, Kathrin Crowell, is seen in dim light, playing with a kitten. In 1874 Eakins and Crowell became engaged; they were still engaged five years later, when Crowell died of meningitis in 1879.

Often this search for individuality required that the subject be painted in his own daily working environment. Eakins' Portrait of Professor Benjamin H. Rand (1874) was a prelude to what many consider his most important work.

In The Gross Clinic (1875), a renowned Philadelphia surgeon, Dr. Samuel D. Gross, is seen presiding over an operation to remove part of a diseased bone from a patient's thigh. Gross lectures in an amphitheater crowded with students at Jefferson Medical College. Eakins spent nearly a year on the painting, again choosing a novel subject, the discipline of modern surgery, in which Philadelphia was in the forefront. He initiated the project and may have had the goal of a grand work befitting a showing at the Centennial Exposition of 1876. Though rejected for the Art Gallery, the painting was shown on the centennial grounds at an exhibit of a U.S. Army Post Hospital. In sharp contrast, another Eakins submission, The Chess Players, was accepted by the Committee and was much admired at the Centennial Exhibition, and critically praised.

In 1876, Eakins completed a portrait of Dr. John Brinton, surgeon of the Philadelphia Hospital, and famed for his Civil War Service. Done in a more informal setting than The Gross Clinic, it was a personal favorite of Eakins, and The Art Journal proclaimed "it is in every respect a more favorable Example of this artist's abilities than his much-talked-of composition representing a dissecting room."

In the 1877 painting William Rush and His Model, he painted the female nude as integral to a historical subject, even though there is no evidence that the model who posed for Rush did so in the nude. The Centennial Exhibition of 1876 helped foster a revival in interest in Colonial America and Eakins participated with an ambitious project employing oil studies, wax and wood Models, and finally the portrait in 1877. william Rush was a celebrated Colonial Sculptor and ship carver, a revered Example of an artist-citizen who figured prominently in Philadelphia civic life, and a founder of the Pennsylvania Academy of Fine Arts where Eakins had started teaching.

Although Eakins was agnostic, he painted The Crucifixion in 1880. Art Historian Akela Reason says

The so-called "Naked Series", which began in 1883, were nude photos of students and professional Models which were taken to show real human anatomy from several specific angles, and were often hung and displayed for study at the school. Later, less regimented poses were taken indoors and out, of men, women, and children, including his wife. The most provocative, and the only ones combining males and females, were nude photos of Eakins and a female model (see below). Although witnesses and chaperones were usually on site, and the poses were mostly traditional in nature, the sheer quantity of the photos and Eakins’ overt display of them may have undermined his standing at the Academy. In all, about eight hundred photographs are now attributed to Eakins and his circle, most of which are figure studies, both clothed and nude, and portraits. No other American Artist of his time matched Eakins' interest in photography, nor produced a comparable body of photographic works.

In 1884, Eakins married Susan Hannah Macdowell, the daughter of a Philadelphia engraver. Two years earlier Eakins' sister Margaret, who had acted as his secretary and personal servant, had died of typhoid. It has been suggested that Eakins married to replace her. Macdowell was 25 when Eakins met her at the Hazeltine Gallery where The Gross Clinic was being exhibited in 1875. Unlike many, she was impressed by the controversial painting and she decided to study with him at the Academy, which she attended for six years, adopting a sober, realistic style similar to her teacher's. Macdowell was an outstanding student and winner of the Mary Smith Prize for the best painting by a matriculating woman Artist.After their childless marriage, she only painted sporadically and spent most of her time supporting her husband's career, entertaining guests and students, and faithfully backing him in his difficult times with the academy, even when some members of her family aligned against Eakins. She and Eakins both shared a passion for photography, both as Photographers and subjects, and employed it as a tool for their art. She also posed nude for many of his photos and took images of him. Both had separate studios in their home. After Eakins' death in 1916, she returned to painting, adding considerably to her output right up to the 1930s, in a style that became warmer, looser, and brighter in tone. She died in 1938. Thirty-five years after her death, in 1973, she had her first one-woman exhibition at the Pennsylvania Academy of Fine Arts.

The forced resignation was a major setback for Eakins. His family was split, with his in-laws siding against him in public dispute. He struggled to protect his name against rumors and false charges, had bouts of ill health, and suffered a humiliation which he felt for the rest of his life. Eakins' popularity amongst the students was such that a number of them broke with the Academy and formed the Art Students' League of Philadelphia (1886–1893), where Eakins subsequently instructed. It was there that he met the student, Samuel Murray, who would become his protege and lifelong friend. He also lectured and taught at a number of other schools, including the Art Students League of New York, the National Academy of Design, Cooper Union, and the Art Students' Guild in Washington DC. Dismissed in March 1895 by the Drexel Institute in Philadelphia for again using a fully nude male model, he gradually withdrew from teaching by 1898.

Of Eakins' later portraits, many took as their subjects women who were friends or students. Unlike most portrayals of women at the time, they are devoid of glamor and idealization. For Portrait of Letitia Wilson Jordan (1888), Eakins painted the sitter wearing the same evening dress in which he had seen her at a party. She is a substantial presence, a vision quite different from the era's fashionable portraiture. So, too, his Portrait of Maud Cook (1895), where the obvious beauty of the subject is noted with "a stark objectivity".

Other outstanding examples of his portraits include The Agnew Clinic (1889), Eakins' most important commission and largest painting, which depicted another eminent American surgeon, Dr. David Hayes Agnew, performing a mastectomy; The Dean's Roll Call (1899), featuring Dr. James W. Holland, and Professor Leslie W. Miller (1901), portraits of educators standing as if addressing an audience; a portrait of Frank Hamilton Cushing (c. 1895), in which the prominent ethnologist is seen performing an incantation at the Zuñi pueblo; Professor Henry A. Rowland (1897), a brilliant scientist whose study of spectroscopy revolutionized his field; Antiquated Music (1900), in which Mrs. william D. Frishmuth is shown seated amidst her collection of musical instruments; and The Concert Singer (1890–92), for which Eakins asked Weda Cook to sing "O rest in the Lord", so that he could study the muscles of her throat and mouth. In order to replicate the proper deployment of a baton, Eakins enlisted an orchestral Conductor to pose for the hand seen in the lower left-hand corner of the painting.

In the late 1890s Eakins returned to the male figure, this time in a more urban setting. Taking the Count (1896), a painting of a prizefight, was his second largest canvas, but not his most successful composition. The same may be said of Wrestlers (1899). More successful was Between Rounds (1899), for which boxer Billy Smith posed seated in his corner at Philadelphia's Arena; in fact, all the principal figures were posed by Models re-enacting what had been an actual fight. Salutat (1898), a frieze-like composition in which the main figure is isolated, "is one of Eakins' finest achievements in figure-painting."

Most notable was his interest in the instruction of all aspects of the human figure, including anatomical study of the human and animal body, and surgical dissection; there were also rigorous courses in the fundamentals of form, and studies in perspective which involved mathematics. As an aid to the study of anatomy, plaster casts were made from dissections, duplicates of which were furnished to students. A similar study was made of the anatomy of horses; acknowledging Eakins' expertise, in 1891 his friend, the Sculptor william Rudolf O'Donovan, asked him to collaborate on the commission to create bronze Equestrian reliefs of Abraham Lincoln and Ulysses S. Grant, for the Soldiers' and Sailors' Arch in Grand Army Plaza in Brooklyn.

Late in life Eakins did experience some recognition. In 1902 he was made a National Academician. In 1914 the sale of a portrait study of D. Hayes Agnew for The Agnew Clinic to Dr. Albert C. Barnes precipitated much publicity when rumors circulated that the selling price was fifty thousand dollars. In fact, Barnes bought the painting for four thousand dollars.

Deeply affected by his dismissal from the Academy, Eakins' later career focused on portraiture, such as his 1905 Portrait of Professor william S. Forbes. His steadfast insistence on his own vision of realism, in addition to his notoriety from his school scandals, combined to hurt his income in later years. Even as he approached these portraits with the skill of a highly trained Anatomist, what is most noteworthy is the intense psychological presence of his sitters. However, it was precisely for this reason that his portraits were often rejected by the sitters or their families. As a result, Eakins came to rely on his friends and family members to model for portraits. His portrait of Walt Whitman (1887–1888) was the poet's favorite.

When he returned to the subject many years later, the narrative became more personal: In William Rush and His Model (1908), gone are the chaperon and detailed interior of the earlier work. The professional distance between Sculptor and model has been eliminated, and the relationship has become intimate. In one version of the painting from that year, the nude is seen from the front, being helped down from the model stand by an Artist who bears a strong resemblance to Eakins.

Eakins was unable to sell many of his works during his lifetime, so when he died in 1916, a large body of artwork passed to his widow, Susan Macdowell Eakins. She carefully preserved it, donating some of the strongest pieces to various museums. When she in turn died in 1938, much of the remaining artistic estate was destroyed or damaged by executors, and the remainders were belatedly salvaged by a former Eakins student. For more details, see the article "List of works by Thomas Eakins".

On October 29, 1917, Robert Henri wrote an open letter to the Art Students League about Eakins:

John Canaday, art critic for The New York Times, wrote in 1964:

In 1982, in his two-volume Eakins biography, art Historian Lloyd Goodrich wrote:

Until recently, major Eakins scholars persistently denied he was homosexual, and such discussion was marginalised. While there is still no consensus, today discussion of homoerotic Desire plays a large role in Eakins scholarship. The discovery of a large trove of Eakins' personal papers in 1984 has also driven reassessment of his life.

Since the 1990s, Eakins has emerged as a major figure in sexuality studies in art history, for both the homoeroticism of his male nudes and for the complexity of his attitudes toward 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 male Models in female classes and vice versa, and was accused of abusing female students.

On November 11, 2006, the Board of Trustees at Thomas Jefferson University agreed to sell The Gross Clinic to the National Gallery of Art in Washington DC, and the Crystal Bridges Museum of American Art in Bentonville, Arkansas for a record $68,000,000, the highest price for an Eakins painting as well as a record price for an individual American-made portrait. On December 21, 2006, a group of donors agreed to match the price in order to keep the painting in Philadelphia. It is displayed alternately at the Philadelphia Museum of Art and at the Pennsylvania Academy of Fine Arts.

Recent scholarship suggests that these scandals were grounded in more than the "puritanical prudery" of his contemporaries—as had once been assumed—and that Eakins' progressive academic principles may have protected unconscious and dubious agendas. These controversies may have been caused by a combination of factors such as the bohemianism of Eakins and his circle (in which students, for Example, sometimes modeled in the nude for each other), the intensity and authority of his teaching style, and Eakins' inclination toward unorthodox or provocative behavior.

He stated his teaching philosophy bluntly, "A Teacher can do very little for a pupil & should only be thankful if he don’t hinder him ... and the greater the master, mostly the less he can say." He believed that women should "assume professional privileges" as would men. Life classes and dissection were segregated but women had access to male Models (who were nude but wore loincloths).