The invention relates to the biological field and discloses an animal source-free and serum-free culture medium of lymphocyte. The culture medium disclosed by the invention essentially consists of IMDM (Iscove Modified Dulbecco Medium), L-glutamine, sodium bicarbonate, recombinant human insulin, human transferrin, human serum albumin, 2-mercaptoethanol, N-acetyl-cysteine, lipid, amino acid, vitamin, microelement, ferric citrate, hydrocortisone, cholamine and non-essential amino acid. The serum-free culture medium disclosed by the invention has the advantages of clear chemical components, no animal source, no serum, safe and ideal culture cells; the instability caused by the doping of animal components and batches is avoided; the result of culturing lymphocyte shows that the total number of the cells and the cell phenotypes are normal; and the serum-free culture medium disclosed by the invention has a good industrial application prospect.