Latrobe attended the Moravian college at Niesky, Saxony, and traveled in France and Italy, acquiring a knowledge of advanced French architecture. After returning to England in 1784, he studied with the Neoclassical architect Samuel Pepys Cockerell. Latrobe may also have studied engineering under John Smeaton, a well-known civil engineer. Having begun his own practice about 1790, Latrobe designed Hammerwood Lodge, Sussex, which shows his subsequent combinations of bold geometric forms with classical details.

Latrobe emigrated in 1795 to the United States, where his first important work was the State Penitentiary in Richmond, Va. (1797–98; demolished 1927). Latrobe then moved to Philadelphia and in 1798 received the commission for his Bank of Pennsylvania, whose Ionic porticoes inspired countless imitations; the building is now considered the first monument of the Greek Revival in America. It is clear, however, that Latrobe did not feel himself confined by styles, as his Sedgeley House, Philadelphia, built about the same time, is thought of as the first Gothic Revival structure in the United States.

In Richmond, Latrobe had met Thomas Jefferson (Jefferson, Thomas), who, in 1803, made him surveyor of the public buildings of the United States. In this post Latrobe inherited the task of completing the U.S. Capitol in Washington, D.C. In the House of Representatives and the Senate chambers, he incorporated American floral motifs—corn cobs, tobacco leaves—into the classical scheme. His Supreme Court Chamber (designed 1806–07) in the Capitol is a notably original American classical interior.

Latrobe's most famous work is the Basilica of the Assumption of the Blessed Virgin Mary, the Roman Catholic cathedral of Baltimore (begun 1805), a severe, beautifully proportioned structure slightly marred by the onion-shaped domes added, after Latrobe's death, to the towers above the portico. Also in Baltimore is his Exchange (1820).

Latrobe was also active as an engineer, especially in the design of waterworks. His more inventive schemes, involving engines, steamboats, and similar projects, brought him to financial ruin. While supervising his waterworks project for New Orleans, Latrobe contracted yellow fever and died. Latrobe set high standards of design and technical competence that were adopted by his foremost pupils, Robert Mills and William Strickland.