Born at Luarca in Spain, Ochoa graduated from Málaga University in 1921 and then proceeded to study medicine at Madrid University, receiving his MD in 1929. Having held research positions in Germany, Spain, and England, he became a research associate in medicine at New York University in 1942, taking American citizenship in 1956. He became a full professor in 1976 and in 1985 was appointed honorary director of the center for molecular biology, University of Madrid.

Ochoa was one of the first to demonstrate the role of high-energy phosphates, e.g., adenosine triphosphate, in the storage and release of the body's energy. While investigating the process of oxidative phosphorylation, in which such triphosphates are formed from diphosphates, he discovered the enzyme polynucleotide phosphorylase. This can catalyze the formation of ribonucleic acid (RNA) from appropriate nucleotides and was later used for the synthesis of artificial RNA. Ochoa was awarded the 1959 Nobel Prize for physiology or medicine for this discovery, sharing the prize with Arthur Kornberg, who synthesized deoxyribonucleic acid (DNA). Ochoa also isolated two enzymes catalyzing certain reactions of the Krebs cycle.