The invention aims at providing a serum-free culture medium for umbilical cord blood mesenchymal stem cells. The serum-free culture medium is composed of a cell factor combination, an alpha-MEM culture solution, insulin, transferring, BMP-4, BOP1, FGF-2 and AMD3100. The cell factor combination comprises IL-3, IL-6, SCF and FL, the concentration of IL-3 is 1-20 ng / ml, the concentration of IL-6 is 1-100 ng / ml, the concentration of SCF is 1-100 ng / ml, and the concentration of FL is 1-100 ng / ml. The serum-free culture medium is developed and researched, the adding sequence of hematopoiesis factors is changed, and the defects of existing umbilical cord blood mesenchymal stem cell culture media in the prior art are overcome.