Tanner quickly accepted the offer. Before the next Salon opened, Tanner set forth for Palestine. Explorations of various mosques and biblical sites as well as character studies of the local population allowed Tanner to further his artistic training. His paintings developed a powerful air of mystery and spirituality. Tanner was not the first Artist to study the Middle East in person. Since the 1830s, interest in Orientalism had been growing in Europe. Artists such as Eugène Delacroix, David Roberts, and later Henri Matisse made such tours to capitalize on this curiosity.

Tanner was born in Pittsburgh, Pennsylvania, the first of seven children. His middle name commemorated the struggle at Osawatomie between pro- and anti-slavery partisans. His father Benjamin Tucker Tanner (1835-1923) was a bishop in the African Methodist Episcopal Church, the first independent black denomination in the United States. Being educated at Avery College and Western Theological Seminary in Pittsburgh, he developed a literary career. In addition, he was a political Activist. His mother Sarah Tanner was born into slavery in Virginia but had escaped to the North via the Underground Railroad. She was mixed race, and Tanner himself was either a quadroon or an octoroon.

Although many artists refused to accept an African-American apprentice, in 1879 Tanner enrolled at the Pennsylvania Academy of the Fine Arts in Philadelphia, becoming the only black student. His decision to attend the school came at an exciting time in the history of artistic institutional training. Art academies had long relied on tired notions of study devoted almost entirely to plaster cast studies and anatomy lectures. This changed drastically with the addition of Thomas Eakins as “Professor of Drawing and Painting” to the Pennsylvania Academy. Eakins encouraged new methods such as study from live Models, direct discussion of anatomy in male and female classes, and dissections of cadavers to further familiarity and understanding of the human body. Eakins’s progressive views and ability to excite and inspire his students would have a profound effect on Tanner. The young Artist proved to be one of Eakins’ favorite students; two decades after Tanner left the Academy, Eakins painted his portrait, making him one of a handful of students to be so honored.

Tanner's Sand Dunes at Sunset, Atlantic City (c. 1885; oil on canvas) hangs in the Green Room at the White House; it is the first painting by an African-American Artist to have been purchased for the permanent collection of the White House. The painting is a landscape with a "view across the cool gray of a shadowed beach to dunes made pink by the late afternoon sunlight. A low haze over the water partially hides the sun." It was bought for $100,000 by the White House Endowment Fund during the Bill Clinton administration from Dr. Rae Alexander-Minter, grandniece of the Artist.

After a photography studio in Atlanta had been unsuccessful, Tanner taught drawing at Clark College which is now called Clark Atlanta University for a short period. In 1891 he traveled to Paris, France, to study at the Académie Julian. He also joined the American Art Students Club of Paris. Paris was a welcome escape for Tanner; within French art circles the issue of race mattered little. Tanner acclimated quickly to Parisian life.

In 1893 on a short return visit to the United States, Tanner painted his most famous work, The Banjo Lesson, while in Philadelphia. The painting shows an elderly black man teaching a boy, assumed to be his grandson, how to play the banjo. This deceptively simple-looking work explores several important themes. Blacks had long been stereotyped as entertainers in American culture, and the image of a black man playing the banjo appears throughout American art of the late 19th century. Thomas Worth, Willy Miller, Walter M. Dunk, Eastman Johnson, and Tanner’s Teacher Thomas Eakins had tackled the subject in their artwork.

Tanner's body of work is not limited to one specific approach to painting and drawing. His works vary from meticulous attention to detail in some paintings to loose, expressive brushstrokes in others. Often both methods are employed simultaneously. The combination of these two techniques makes for a masterful balance of skillful precision and powerful expression. Tanner was also interested in the effects that color could have in a painting. Many of his paintings accentuate a specific area of the color spectrum. Warmer compositions such as The Resurrection of Lazarus (1896) and The Annunciation (1898) express the intensity and fire of religious moments, and the elation of transcendence between the Divine and humanity. Other paintings emphasize cooler, blue hues. Works such as The Good Shepherd (1903) and Return of the Holy Women (1904) evoke a feeling of somber religiosity and introspection. Tanner often experimented with light in a composition. The source and intensity of light and Shadow in his paintings create a physical, almost tangible space and atmosphere while adding emotion and mood to the environment. Tanner also used light to add symbolic meaning to his paintings. In The Annunciation (1898) the angel Gabriel is represented as a column of light that forms, together with the shelf in the upper left corner, a cross. This view of the representation of Gabriel is consistent with James Romaine's comment that "Through the visual language of her pose and expression Tanner draws the viewer into Mary’s inner life of virtue, trepidation, acceptance, and wonderment." Mary's acceptance includes her acceptance of the cross that she will have to bear by consenting to be the Lord's handmaid (Luke 1:38).

In 1899 he married Jessie Olsson, a white Swedish-American opera singer. A contemporary, Virginia Walker Course, described their relationship as one of equal talents, but racist attitudes insisted the relationship was unequal: "Fan, did you ever hear of a miss [sic] Olsson of Portland? She has a beautiful voice I believe and came to Paris to cultivate it and she has married a darkey Artist [...] He is an awefully [sic] talented man but he is black. [...] She seems like a well educated girl and really very nice but it makes me sick to see a cultivated woman marry a man like that. I don't know his work but he is very talented they say." Jessie Tanner died in 1925, twelve years before her husband, and he grieved her deeply through the Twenties. He sold the family home in Les Charmes where they had been so happy together. They are buried next to each other in Sceaux, Hauts-de-Seine. They had a son Jesse, who survived Tanner at his death.

During World War I, Tanner worked for the Red Cross Public Information Department, at which time he also painted images from the front lines of the war. His works featuring African-American troops were rare during the war. In 1923 the French state made him a knight of the Legion of Honour for his work as an Artist.

Tanner met with fellow African-American Artist Palmer Hayden in Paris circa 1927. They discussed artistic technique and he gave Hayden advice on interacting with French society.

Tanner died peacefully at his home in Paris, France, on May 25, 1937.

Tanner's work was influential during his career; he has been called "the greatest African American Painter to date." The early paintings of william Edouard Scott, who studied with Tanner in France, show the influence of Tanner’s technique. In addition, some of Norman Rockwell’s illustrations deal with the same themes and compositions that Tanner pursued. Rockwell's proposed cover of the Literary Digest in 1922, for Example, shows an older black man playing the banjo for his grandson. The light sources are nearly identical to those in Tanner's Banjo Lesson. A fireplace illuminates the right side of the picture, while natural light enters from the left. Both use similar objects as well such as the clothing, chair, crumpled hat on the floor.