The invention discloses a separation and culture method of 
umbilical cord blood derived mesenchymal stem cells, and belongs to the technical field of 
biology. The separation and culture method comprises the following steps of performing centrifuging treatment on 
umbilical cord blood, taking an upper layer to obtain 
plasma, and taking a lower layer to obtain cells; performing inactivation treatmenton the 
plasma, then performing centrifuging treatment, and taking supernatant so as to obtain autoserum; mixing the lower-layer cells with normal 
saline to obtain a mixture, then placing the mixturein 
lymphocyte separation liquid, and performing centrifuging treatment so as to obtain 
umbilical cord blood mononuclear cells; and re-suspending the umbilical 
cord blood mononuclear cells with serum-free 
culture mediums, placing the re-suspended umbilical 
cord blood mononuclear cells in an autoserum 
coating culture dish, performing 
primary culture to obtain primary mesenchymal stem cells, adding 
cell dissociation liquid into the primary mesenchymal stem cells, performing 
cell dissociation treatment, and then performing subculturing. According to the method, penicllin-
streptomycin for 
cell culture is not added in a 
separation process and a culture process, so that 
toxicity is not generated on the cells. In addition, according to the method, the autoserum 
coating culture dish for centrifugalseparation is adopted, so that animal derived viruses cannot be introduced.