The invention discloses a hybridoma cell serum-free medium, which comprises the following raw materials: alanine, arginine, aspartic acid, asparagine, cystine, cysteine, glutamine, glutamic acid Amino acid, glycine, histidine, etc., calcium chloride, magnesium sulfate, potassium chloride, potassium dihydrogen phosphate, disodium hydrogen phosphate, sodium bicarbonate, sodium hydroxide, ferrous sulfate heptahydrate, magnesium chloride hexahydrate, sulfuric acid Copper, sodium pyruvate, manganese dichloride, sodium selenite, ammonium molybdate, etc., nicotinamide, p-aminobenzoic acid, folic acid, ribose, uracil, lipoic acid, linoleic acid L, cholesterol, dexamethasone, Estradiol progesterone, testosterone, etc. The medium of the present invention does not contain serum components at all; the purity of the produced antibody is greatly improved; the medium has high amplification speed and high antibody expression ability; the medium can maintain cell activity for a long time, and the hybridoma cell culture time is greatly improved Extending can greatly extend the production time and increase the antibody production.