Har Khorana was born in Raipur, West Pakistan. His father was a clerk in the British Indian government. Although the family was not well-to-do, Khorana's father made sure that his children had an education.

Khorana went to Punjab University in Lahore and graduated with a Master of Science. In 1945, a fellowship from the government of India gave him the opportunity to study abroad. He went to the University of Liverpool where he obtained his doctorate.

Khorana spent the next few years doing post-doctorate work, first at the Eidgen�ssische Technische Hochschule in Zurich, then at Cambridge University with G. W. Kenner and Lord Alexander R. Todd. It was at Cambridge that Khorana developed an interest in proteins and nucleic acids.

In 1952, Khorana was offered a job at the University of British Columbia where he was able to work fairly independently on various research projects involving phosphate esters and nucleic acids. The work on the chemical synthesis of ribotrinucleotides for protein synthesis was initiated at this time.

In 1960, Khorana accepted a position in the Institute for Enzyme Research at the University of Wisconsin. He continued working on nucleotide synthesis and cracking the genetic code. For this work Khorana shared the 1968 Nobel Prize in Physiology or Medicine with Robert Holley and Marshall Nirenberg.

Since 1970, Khorana has been the Alfred P. Sloan Professor of Biology and Chemistry at the Massachusetts Institute of Technology.