The invention provides a lymphocyte serum-free medium which comprises the following materials per liter: a basic culture medium, 10 mg of transferrin, 0.01 mg of sodium selenite, 110 mg of pyroracemic acid, 1.5 mg of ethanolamine, 10 mg of insulin, 10 to 30 mg of glutamine substitute, 300 mg of lipid-type bovine serum albumin, 0.001 mg of cupper sulfate, 0.0005 mg of ammonium meta-vanadate, 0.00005 mg of manganese chloride, 0.8 mg of zinc sulfate, 2 mg of Vitamin E, 100 mg of PHA, and 10 to 25 mg of an MHEPES/NaHCO3 buffering system. The invention further provides a preparation method and application to lymphocyte culture of the lymphocyte serum-free medium. The lymphocyte serum-free medium has the advantages that the repeatability of lymphocyte culture is high; poisoning and polluting of blood serum to lymphocyte are avoided; convenience is brought for differentiation of the lymphocyte, so as to obtain various mitosis phases.