Educated at the École Normale Supérieure, Paris, Perrin joined the faculty of the University of Paris (1898) where he became professor of physical chemistry (1910–40). In 1895 he established that cathode rays are negatively charged particles (electrons). His attempt to determine the mass of these particles was soon anticipated by the work of J.J. Thomson.

About 1908 Perrin began to study Brownian motion, the erratic movement of particles suspended in a liquid. Einstein's mathematical analysis (1905) of this phenomenon suggested that the particles were being jostled by the randomly moving water molecules around them. Using the newly developed ultramicroscope, Perrin carefully observed the manner of sedimentation of these particles and provided experimental confirmation of Einstein's equations. His observations also enabled him to estimate the size of water molecules and atoms as well as their quantity in a given value. This was the first time the size of atoms and molecules could be reliably calculated from actual visual observations. Perrin's work helped raise atoms from the status of useful hypothetical objects to observable entities whose reality could no longer be denied.