The invention belongs to the field of detection, and particularly relates to a serum-free medium capable of amplifying mesenchymal stem cells and a preparation method thereof. The serum-free medium mainly contains curcumin, cepharanthine, berberine, resveratrol, anthocyanidin, bone morphological protein 4, beta-glycerophosphate, cholesterol, isobutyl methyl xanthine, EGF, biotin, aFGF, heparin sodium, reduced glutathione, transferrin, insulin, ascorbic acid, dexamethasone, interleukin 2, vitamin B12, interleukin 6, stem cell factors, nicotinamide, TGF-beta 3, thrombopoietin, taurine, rosiglitazone, FMS-like tyrosine kinase 3 ligand, IGF2, sodium pyruvate, GM-CSF, TPO and the like. The serum-free culture medium not only has the capacity of amplifying mesenchymal stem cells derived from fat, placenta, bone marrow and umbilical cord in vitro, but also can maintain self-renewal of the stem cells, prevent differentiation of the stem cells and inhibit canceration of the stem cells.