The invention relates to the technical field of cell culture, and in particular to a serum-free culture medium, a preparation method thereof and a culture method of mesenchymal stem cells. The culturemedium consists of the following components: non-essential amino acid, glutamine, hydrocortisone, dexamethasone, polyvinyl alcohol, recombinant human insulin, recombinant human transferrin, a recombinant human epidermal growth factor, a recombinant human basic fibroblast growth factor, a recombinant human Wnt-3a protein, a recombinant human fibronectin, recombinant human laminin, L-glutathione, L-ascorbic acid, beta-mercaptoethanol, ethanolamine, Pluronic F-68, Tween 80, cholesterol, adenine, sodium selenite and a basic culture medium. According to the invention, polyvinyl alcohol can replaceserum albumin in a serum-free culture medium in carrier function, so that proliferation capacity of mesenchymal stem cells is effectively improved. Polyvinyl alcohol has more stable performance thanserum albumin, and the problem that proliferation capacity and passage capacity of stem cells are reduced can be effectively solved.