The present study aimed to establish an efficient and stable separation method of Potentilla anserina callus protoplast, which were induced from root tuber of Qinghai Juema No.4. The design method of L₉(3⁴) orthogonal experimental was adopted to optimize the combination conditions. The effects of some factors on the separation of Potentilla anserina callus protoplast were investigated, including enzyme combination, digestiontime, mannitol concentration and centrifugal speeds. The results indicated that the sequential order of every factor for protoplast yield was enzyme combination, digestion time, mannitol concentration and centrifugal speeds.The optimal enzyme solution for protoplast isolation was 2.0% cellulase R10 + 0.75% Pectinase Y23 + 0.5 mol/L mannitol.The digestion was conducted on constanttemperature shaker with 40 r/min at 25 ℃ for 10h in dark and centrifugation with 1 000 r/min for 6 min for protoplast collection.The protoplast yield amounted to 8.96×10⁵ cells/g and the vitality was up to 92.77%.