Steen was enrolled at the University of Leiden in 1646 and in 1648 was one of the founding members of the Leiden painters' Guild of St. Luke. His early teachers seem to have been the historical painter Nicolaus Knupfer at Utrecht, genre and landscape painter Adriaen van Ostade at Haarlem, and the landscapist Jan van Goyen at The Hague. In 1649 Steen married van Goyen's daughter and settled at The Hague for the next few years. He moved to Delft in 1654 and to Haarlem in 1661. In 1670 he was back in Leiden, and in 1673 he married again.

In Steen's landscapes, including his winter scenes, small earthy figures recall those of Adriaen and of Isack van Ostade. In his later works the figures are larger, less crowded, and more individually characterized. He shows them playing cards or skittles, or carousing in their cups, as in “Skittle Players Outside an Inn” (c. 1660; National Gallery, London). His frequent use of inns probably reflects his own background as the son of a brewer and sometime brewer and tavernkeeper himself. He was a master at capturing subtleties of facial expression, especially in children. His best works display great technical skill, particularly in the handling of colour.

During Steen's last years, his paintings began to anticipate the Rococo style of the 18th century, becoming increasingly elegant and somewhat less energetic, as in the “Serenade” (c. 1675; Národní Galerie, Prague), and showing a heavy French influence and an increased flamboyance.