The present invention provides a cryopreservation method of tobacco leaf callus cells, comprising the steps of: culturing the tobacco leaf callus cells in a medium to prepare a cell suspension; centrifuging the cell suspension and removing supernatant to get a cell solution, transferring the cell solution to a cryopreservation tube, adding a cryoprotectant therein, then sealing, cooling the cryopreservation tube to 4 DEG C and standing at the condition for 20-40 minutes, continue cooling to -20 DEG C and standing for 30-40 minutes at the temperature, continue cooling to -40 DEG C and standing for 3-4 hours at the temperature, and last storing the cryopreservation tube at -80 DEG C. The method provided by the invention is simple and easy, the cryopreserved cells have high survival rate, and the method does not consume a large amount of manpower and resources and is low in cost.