Although born in Nicholaev, Russia, Breit moved to America in 1915 and became a naturalized citizen in 1918. He studied at Johns Hopkins University, gaining his PhD in 1921. From 1921 until 1924 he worked successively at the universities of Leiden, Harvard, and Minnesota, before joining the Carnegie Institution, Washington (1924–29).

At Carnegie, Breit worked in the department of terrestrial magnetism as a mathematical physicist, and it was there that he conducted, with Merle A. Tuve, some of the earliest experiments to measure the height and density of the ionosphere. Their technique was to transmit short bursts of radio waves and analyze the reflected waves received. Their work is now seen as a significant step in the historical development of radar.

Besides his pioneering studies of the ionosphere, Breit also worked on quantum theory, nuclear physics, and quantum electrodynamics. In particular, he and Eugene Wigner were able to show that the experimental observations of the interactions of neutrons and protons indicated that the particles differed only in their charge and other electrical properties, and not in their nuclear forces. The Breit–Wigner formula is a formula for the energy dependence of the absorption cross-section of a compound nucleus in a nuclear reaction.

Between 1929 and 1973 Breit held professorial posts at the universities of New York, Wisconsin, and Yale, and the State University of New York, Buffalo.