The invention relates to the field of separation culture of dental pulp stem cells, and discloses a separation culture method of the dental pulp stem cells. The method comprises the following steps: crushing a tooth to expose dental pulp tissues, cutting the dental pulp tissues into pieces, collecting into a centrifuge tube, and using collagenase type I and neutral protease to decompose the dental pulp tissues; adding a complete culture solution to finish the decomposition, centrifuging to remove supernatant, then resuspending cells by using the complete culture solution, and filtering to obtain primary cell suspension liquid; adopting 1*104/cm<2> as primary cell planting density; two days later, changing the liquid for the first time, then changing the liquid for once every three days, culturing for 9-19 days, wherein about 100 clone cells are formed by each primary cell, and carrying out cell harvesting and passage; planting passage cells according to the planting density of 1*4000/cm<2>, and carrying out cell harvesting and passage again when the cells reach 70-90% of convergence degree. The separation culture method increases the separation culture efficiency and yield of the dental pulp stem cells and reduces the production cost by regulating the separation culture method of the dental pulp stem cells and all parameters in the concrete operation.