Born the son of a magistrate in Palermo, Sicily, Cannizzaro studied physiology in his native city and at Naples. He turned to organic chemistry after realizing the importance of chemical processes in neurophysiology, and from 1845 to 1847 worked as a laboratory assistant to R. Piria at Pisa. Cannizzaro was an ardent liberal and in 1847 he returned to Sicily to fight as an officer in the insurrection against the ruling Bourbon regime. Following the abortive revolution of 1848 he went into exile and returned to chemistry, working with Michel Eugène Chevreul in Paris (1849–51).

Cannizzaro returned to Italy in 1851 as professor of chemistry and physics at the Collegio Nazionale at Alessandria. In 1853 he discovered the reaction known as Cannizzaro's reaction, in which an aromatic aldehyde is simultaneously oxidized and reduced in the presence of concentrated alkali to give an acid and an alcohol.

In 1855 Cannizzaro moved to Genoa as professor of chemistry and here he produced the work for which he is chiefly remembered. His pamphlet Sunto di un corso di filosofia chimica (1858; Epitome of a Course of Chemical Philosophy) finally resolved more than 50 years of confusion about atomic weights. In 1860 a conference was held at Karlsruhe, Germany, to discuss the problem. No agreement was reached but Cannizzaro's pamphlet was circulated and soon after was widely accepted. In it Cannizzaro restated the hypothesis first put forward by Amedeo Avogadro, clearly defined atoms and molecules, and showed that molecular weights could be determined from vapor-density measurements.

Politics intervened once more in Cannizzaro's life and in the struggle to reunite Italy he returned to Palermo in 1860 to join Garibaldi. He was professor of inorganic and organic chemistry at Palermo until 1870, when he went to Rome to found the Italian Institute of Chemistry. The most notable research of this last period was that on santonin, a compound derived from species of Artemisia (wormwoods) that is active against intestinal worms, which Cannizzaro showed to be a derivative of naphthalene. He was widely honored and became a senator in 1871.