After a stint in the Italian Army from 1928 and 1929, he worked with Otto Stern in Hamburg and Pieter Zeeman in Amsterdam as a Rockefeller Foundation fellow in 1930. Segre was appointed assistant professor of physics at the University of Rome in 1932 and served until 1936. From 1936 to 1938 he was Director of the Physics Laboratory at the University of Palermo. There, he discovered technetium, the first artificially synthesized chemical element which does not naturally occur.
Segre, as a Jew, was dismissed from the University of Palermo by Italy's Fascist government on a 1938 visit to California, so he stayed in the U.S. as a research associate in the Berkeley Radiation Laboratory and a lecturer of the physics department at the University of California, Berkeley. At Berkeley, he helped discover the element astatine and the isoptope plutonium-239 (which was later used to make the atom bomb dropped on Nagasaki).
From 1943 to 1946 he worked at the Los Alamos National Laboratory as a group leader for the Manhattan Project. He was naturalized a U.S. citizen in 1944. Upon his return to Berkeley in 1946, he became a professor of physics, serving until 1972. In 1974, he returned to the University of Rome as a professor of nuclear physics.
He died at the age of 84 of a heart attack.