Ochoa was born in Luarca (Asturias), Spain. His father was Severo Manuel Ochoa, (who he was named after), a lawyer and businessman,and his mother was Carmen de Albornoz.

His father died when Ochoa was seven, and he and his mother moved to Málaga, where he attended elementary school through high school.

Ochoa's interest in biology was stimulated by the publications of the Spanish neurologist and Nobel laureate Santiago Ramón y Cajal.

In 1923, he went to the University of Madrid Medical School, where he hoped to work with Cajal, but Cajal retired. He studied with father Pedro Arrupe, and Juan Negrín was his teacher.

Negrín encouraged Ochoa and another student, José Valdecasas, to isolate creatinine from urine. The two students succeeded and also developed a method to measure small levels of muscle creatinine.

Ochoa spent the summer of 1927 in Glasgow working with D. Noel Paton on creatine metabolism improving his English skills.

During the summer he refined the assay procedure further and upon returning to Spain he and Valdecasas submitted a paper describing the work to the Journal of Biological Chemistry, where it was rapidly accepted, marking the beginning of Ochoa's biochemistry career.

Ochoa completed his undergraduate medical degree in the summer of 1929 and developed an interest in going abroad to gain further research experience. His previous creatine and creatinine work led to an invitation to join Otto Meyerhof's laboratory at the Kaiser Wilhelm Institute for Biology in Berlin-Dahlem in 1929.

In 1930 Ochoa returned to Madrid to complete research for his MD thesis, which he defended that year, and minted a new MD in 1931.

Ochoa married Carmen García Cobián, he did not have any children.

He then began postdoctoral study at the London National Institute for Medical Research (NIMR), where he worked with Henry Hallett Dale. His London research involved the enzyme glyoxalase and was an important departure in Ochoa's career in two respects. First, the work marked the beginning of Ochoa's lifelong interest in enzymes. Second, the project was at the cutting edge of the rapidly evolving study of intermediary metabolism.

In 1933 the Ochoas returned to Madrid where he began to study glycolysis in heart muscle.

Within two years, he was offered the directorship of the Physiology Section in a newly created Institute for Medical Research at the University of Madrid Medical School. Unfortunately the appointment was made just as the Spanish Civil War erupted. Ochoa decided that trying to perform research in such an environment would destroy forever his "chances of becoming a scientist." Thus, "after much thought, my wife and I decided to leave Spain."

In September 1936 Severo and Carmen began what he later called the "wander years" as they traveled from Spain to Germany, to England, and ultimately to the United States within a span of four years.

Ochoa left Spain and returned to Meyerhof's Kaiser Wilhelm Institute for Biology now relocated in Heidelberg, where Ochoa found a profoundly changed research focus. By 1936 Meyerhof's laboratory had become one of the world's foremost biochemical facilities focused on processes such as glycolysis and fermentation. Rather than studying muscles "twitch," the lab was now purifying and characterizing the enzymes involved in muscle action but were involved in yeast fermentation.

From 1938 until 1941 he was Demonstrator and Nuffield Research Assistant Ocho at the University of Oxford.

Ochoa then went to the United States, where he again held many positions at several universities. Between 1940 and 1942, Ochoa worked for the Faculty of Medicine of Washington University in St. Louis.

In 1942 he was appointed Research Associate in Medicine at the New York University School of Medicine and there subsequently became Assistant Professor of Biochemistry (1945), Professor of Pharmacology (1946), Professor of Biochemistry (1954), and Chairman of the Department of Biochemistry.

In 1959, Ochoa and Arthur Kornberg were awarded the Nobel Prize for Physiology or Medicine "for their discovery of the mechanisms in the biological synthesis of ribonucleic acid and deoxyribonucleic acid".

Ochoa received the U.S. National Medal of Science in 1978.

Ochoa continued research on protein synthesis and replication of RNA viruses until 1985, when he returned to Spain and gave advice to Spanish science policy authorities.

Severo Ochoa died in Madrid, Spain on 1 November 1993.