Chassériau was born in Samaná, in Saint Domingue (now the Dominican Republic). His father was a French adventurer who, at the time of Théodore's birth, held an administrative position in what was then a French colony; his mother was the daughter of a Creole landowner. The family moved to Paris in 1821, where the young Chassériau soon showed precocious drawing skill. He was accepted into the studio of Jean-Auguste-Dominique Ingres in 1830, at the age of eleven, becoming the favorite pupil of the great classicist, who came to regard him as his truest disciple.
After Ingres left Paris in 1834 to become director of the French Academy in Rome, Chassériau fell under the influence of Eugène Delacroix, whose brand of painterly colorism was anathema to Ingres. Chassériau's art has often been characterized as an attempt to reconcile the classicism of Ingres with the romanticism of Delacroix. He first exhibited at the Paris Salon in 1836, and was awarded a third-place medal in the category of history painting. In 1840 Chassériau travelled to Rome and met with Ingres, whose bitterness at the direction his student's work was taking led to a decisive break.
Throughout his life he was a prolific draftsman; his many portrait drawings executed with a finely pointed graphite pencil are close in style to those of Ingres. He also created a body of 29 prints, including a group of eighteen etchings of subjects from Shakespeare's "Othello" in 1844. In 1846, Chassériau made his first trip to Algeria. From sketches made on this and subsequent trips he painted such subjects as "Arab Chiefs Visiting Their Vassals" and "Jewish Women on a Balcony". A major late work, "The Tepidarium", depicts a large group of women drying themselves after bathing, in an architectural setting inspired by the artist's trip in 1840 to Pompeii. His most monumental work was his decoration of the grand staircase of the Cour des Comptes, commissioned by the state in 1844 and completed in 1848. This work was heavily damaged in May 1871 by a fire set during the Commune, and only fragments could be recovered; these are preserved in the Louvre.
After a period of ill health, exacerbated by his exhausting work on commissions for murals to decorate the Churches of Saint-Roch and Saint-Philippe-du-Roule, Chassériau died at the age of 37 in Paris, on October 8, 1856.
His work had a significant impact on the style of Puvis de Chavannes and Gustave Moreau, and (through those artists' influence) reverberations in the work of Paul Gauguin and Henri Matisse.