Abstract: [Objective] The present study aim to establish an efficient and stable separation method of Lilium pumilum DC. callus protoplast, to provide scientific basis for Lilium pumilum DC. breeding research. [Method]The design method of L16(3⁴) orthogonal experimental was adopted to optimize the combination conditions. The effects of some factors on the separation of Lilium pumilum DC. callus protoplast was investigated, including enzymolysis time, enzyme combination, osmotic pressure, pretreatment,and centrifugal speeds.[Result] (1)Pretreatment was carried out under the conditions of 0.015 g/mL cellulase + 0.005 g/mL pectinase + 0.65 mol/L mannitol. The results showed that the optimal hydrolysis time was 6 h and the yield was 3.47×106 cells/mL.(2) According to the results of the orthogonal test ,the optimum condition for protoplast isolation from Lilium pumilum DC. included a mixed enzyme solution of 0.02 g/mL cellulase、0.008 g/mL pectolyase、0.6 mol/L mannitol. High activity and pure protoplasts could be prepared by using the optimum condition, protoplast yield up to 3.465×106 cells/mL, viability of 60.32%;(3) Under the optimal enzyme combination treatment,Under these conditions, the dark matter treatment could increase the protoplast survival rate to 76.15%; At 15 min and 1000 r/min, the protoplast yield was the highest at 3.51×106/mL; at 10 min and 800 r/min, the protoplast activity was the highest at 61.2%.[Conclusion]The experiment obtained the best separation conditions of protoplasts of Lilium pumilum DC. and established a protoplast isolation system, which laid a foundation for further research on Lilium pumilum DC. breeding.