Daulat Singh Kothari had his early education at Udaipur and Indore and received a master's degree in physics from Allahabad University in 1928 under guidance of Meghnad Saha. For his Doctor of Philosophy, Kothari worked at the Cavendish Laboratory, University of Cambridge under the supervision of Ernest Rutherford, to whom he was recommended by Meghnad Saha.
After his return to India, Daulat Singh Kothari worked at the Kaiden University from 1934 to 1961 in various capacities as reader, professor and Head of the Department of Physics. He was scientific advisor to Ministry of Defence from 1948 to 1961 and was then appointed as chairman of the University Grants Commission in 1961 where he worked till 1973. He was the chairman of the Indian Education Commission of 1964–1966, popularly known as Kothari Commission, which was the first ad hoc commission set up in India for the modernization and standardization of education in India.
Daulat Singh Kothari was a member of Indian Science Congress (Past-President) Association of Scientific Workers (Vice-President) & various India Committees.