The invention discloses a human umbilical cord mesenchymal stem cell separation and culture method. The culture method is characterized by comprising the following steps of sample collection and receiving, cell separation, primary cell culture, subculture and cell detection. The human umbilical cord mesenchymal stem cell separation and culture method has the advantages that the cell culture periodis greatly shortened, and a large quantity of mesenchymal stem cells with high purity and a good state are quickly obtained. By means of the separation and culture method, Wharton's jelly is separated, besides, two adventitias are stripped, the growth state of the obtained hUC-MSCs is good, the number of obtained P1 generation cells can reach (3-12.5)*10<7>, surface antibodies CD44+ and CD105+ can reach (99.6+/-0.2)% through flow-type detection, and the method has good adipogenesis and osteogenesis differentiation capacity.