Ponnamperuma was born in Gale, Sri Lanka, an island near India formerly called Ceylon. He was educated at the University of Madras in India and received his B.A. in 1948. He then went on to the University of London where he earned a B.S. in 1959. Following this, he attended the University of California at Berkeley to pursue a doctorate in chemistry, which he earned in 1962. Four years later he became an American citizen.

Ponnamperuma worked as a research assistant at Lawrence Radiation Lab at the University of California at Berkeley. In 1963 he went on to work as a scientist at Ames Research Center, a facility funded by the National Aeronautics and Space Administration (NASA). Three years later, he took a position at the University of Maryland working as professor of chemistry and serving as Director of the Lab of Chemical Evolution.

Ponnamperuma's research at the University of Maryland helped him become one of the world's most noted scientific researchers investigating the origins of life. He furthered important theories set forth by renowned scientists Aleksandr Oparin, Stanley Miller and Harold Clayton Urey. Ponnamperuma focused on producing compounds related to the nucleic acids, and demonstrated that nucleotides and dinucleotides can be formed by random processes alone. In another achievement, he showed the formation of ATP, a compound critical to the use of energy within a cell.

Ponnamperuma held various positions throughout his career, including that of principal investigator for the NASA Apollo Program and director to UNESCO's Institute of Early Education. He was also a foreign member of the Indian National Science Academy and was awarded an honorary degree from the University of Sri Lanka. In 1989 he established the Third World Foundation of North America, an organization devoted to scientific and governmental collaboration between undeveloped and developed countries.