The invention discloses a preparation method of deciduous tooth mesenchymal stem cells, comprising the steps of: cleaning and sterilizing the tissue surfaces of deciduous teeth with normal saline and 75% alcohol, preserving the deciduous teeth in deciduous teeth preserving fluid; acquiring dental pulp from the deciduous teeth, adding dental pulp digestive fluid to the dental pulp for digestion; obtaining a unicell suspension; adding cell culture fluid, carrying out centrifuging and cleaning, adding precipitated cells into cell culture fluid, putting the cell culture fluid in a carbon dioxide incubator for culturing; when the primary cell fusion reaches 70%, adding cell washing fluid, shaking the culture bottle for washing cells, sucking and abandoning the cell washing fluid, adding cell digestive fluid for digestion, adding cell culture fluid to terminate digestion, repeatedly beating upon the bottle bottom until the cells completely fall off, adding cell culture fluid, inoculating into a culture bottle, putting the culture bottle in the carbon dioxide incubator for culturing, regarding the cells as P1 generation mesenchymal stem cells; when the P1 generation cell fusion reaches 70-80%, carrying out trypsin digestion, collecting digestive cells, carrying out centrifuging and inoculation; after 3d, carrying out trypsin digestion again, collecting digestive cells, carrying out centrifuging, counting and inoculation, and when the P3 generation cell growth reaches 80%, gathering and cryopreserving the cells.