Born at Leiden on 7 April 1613, the son of a glass-engraver. He was apprenticed to an engraver and then to a glass-engraver before entering the glaziers' guild in 1625. On 24 February 1628 he entered Rembrandt's studio, remaining there probably until his master's removal to Amsterdam in 1631/2. He then became an independent master. In 1648 he was a founder member of the Leiden guild.

He painted genre and history subjects, portraits and still lifes in a meticulous style which was lavishly rewarded by noble patrons who included Christina of Sweden, Archduke Leopold Wilhelm of Austria and Charles II of England (whose invitation to London Dou is said to have refused). He was the founder of the school known as the Fijnschilders ('fine painters') whose influence lasted into the nineteenth century. His whole life was spent in Leiden, where he owned four houses, and where he was buried in February 1675.