The invention relates to the field of biology, and discloses a serum-free culture medium which essentially comprises an IMDM (Iscove Modified Dulbecco Medium), L-glutamine, sodium bicarbonate, Hepes, recombinant human insulin, recombinant human transferrin, recombinant human albumin, 2-mercaptoethanol, protocatechuic acid, lipid, amino acid, vitamins, trace elements, Pluronic F-68, hydrocortisone, vitamin C, bonding amine or recombinant human fibronectin, progesterone, putrescine, heparin, serotonin, epidermal growth factors (EGFs), b-fibroblast growth factors (FGF), platelet derive growth factor (PDGF)-BB and insulin-like growth factor (IGF)-I. The serum-free culture medium is clear in chemical components, free from animal sources and serum and safe and ideal in cell cultivation and avoids the doped animal components and unstable batches, and the results of the cultured mesenchymal stem cells show that the total cellular score, the cell phenotype and the secretory cell factors are normal, so that the serum-free culture medium has good industrial application prospect.