Avogadro was born in Turin in northern Italy and came from a long line of lawyers. He too was trained in law and practiced for some years before taking up the study of mathematics and physics in 1800. His early work was carried out in the field of electricity, and in 1809 he became professor of physics at the Royal College at Vercelli. He was professor of mathematical physics at Turin from 1820 until 1822 and from 1834 to 1850.

His fame rests on his paper Essai d`une manière de determiner les masses relatives des molecules des corps et les proportions selon lesquelles entrent dans cet combinaisons (1811; On a Way of Finding the Relative Masses of Molecules and the Proportions in which They Combine), published in the Journal de Physique. This states the famous hypothesis that equal volumes of gases at the same temperature and pressure contain equal numbers of molecules. It follows from the hypothesis that relative molecular weights can be obtained from vapor densities and that the proportion by volume in which gases combine reflects the combining ratio of the molecules. Using this theory, Avogadro showed that simple gases such as hydrogen and oxygen are diatomic (H₂, O₂) and assigned the formula H₂O to water, whereas John Dalton had arbitrarily assumed that the simplest compound of two elements would have the formula HO.

Avogadro's work provided the essential link between Gay-Lussac's law of combining volumes and Dalton's atomic theory. This was not, however, realized at the time and, as a consequence, the determination of a self-consistent set of atomic weights was delayed for 50 years. The French physicist André Ampère was one of the few who accepted the theory and for many years it was taken to be Ampère's own.

Avogadro's contribution to chemistry was not appreciated in his own lifetime. The importance and truth of the theory was unrecognized until 1860 when his fellow Italian, Stanislao Cannizzaro, forcefully restated it at the Karlsruhe Conference and demonstrated that it was the key needed to unlock the problem of atomic and molecular weights. The number of particles in one mole of a substance was named Avogadro's constant or number in his honor. It is equal to 6.022 52 × 10²³.