Born in North Kingstown, Rhode Island, he grew up in Newport and was tutored by Cosmo Alexander, Scottish painter living there, later relocating with Alexander to Scotland to finish his studies. His mentor died in Edinburgh in 1771, and after briefly trying to earn a living at his art, Stuart returned to Newport in 1772.
Stuart left for England in 1775 to pursue what became his second unsuccessful attempt at a vocation in painting, becoming after a time a protege of Benjamin West, with whom he studied for the following six years. The relationship was a successful one, and Stuart was exhibiting at the Royal Academy by 1777.
By 1782, Stuart's third attempt to make a living at his art met with considerable success, largely based on the acclaim from his "Portrait of a Gentleman Skating." Despite the number of commissions, however, Stuart's naïvete with regard to finances soon led to his falling into considerable debt, for which he was threatened with debtors' prison. He therefore fled to Ireland in 1787, where he painted with equal success and managed his money equally miserably.
Stuart returned to the United States in 1793, settling briefly in New York City and relocating the next year to Philadelphia, where he opened a studio. It was here he would gain not only a foothold in the art world but lasting fame, executing portrait paintings of many of the most important Americans of the day, including several of George Washington of which the most famous is probably the "Athenaeum Head" currently on the dollar bill.
In 1803 Stuart opened a studio in Washington, D. C where he was in an optimum position to paint portraits of presidents and other major personages. By the end of his career, he had painted over a thousand portraits of American political figures.
He moved to Boston in 1805, continuing his history of critical acclaim and financial troubles. In 1825 he began to show signs of paralysis, but continued to paint. Stuart died in Boston at the age of 72 and is buried there in the Old South Burial Ground.