【Objective】The present experiment was conducted to study the optimal conditions for isolation of protoplast from young leaves of papaya. 【Method】The young leaves of papaya were sampled from virus-free plantlet to isolate the protoplast， and the influences of different conditions including enzymolysis solution combinations （1-3% cellulase and 0.5-0.8% pectinase）， incubation times （0-12 h）， purification centrifugal speed （500-1500 rpm） and centrifugal time （6-10 min） on isolation of protoplasts were compared and analyzed. 【Result】The quantity of purified protoplast increased gradually with the increasing concentration of cellulase and pectinase enzymes in enzymolysis solution， while its activity showed declining trend. The best results were obtained by incubating leaf material for 8 h in solution containing combination of 2.0% cellulase +0.5% pectolyase. Increasing the mannitol concentration in enzymolysis solution，the quantity of protoplasts initially increased but then showed a declining trend， and gave highest output （2.48×106 protoplast/gFW） at 0.55 mol/L mannitol concentration in enzymolysis solution. The best purification was obtained at 1000 rpm centrifuge speed for 6-8 min. 【Conclusion】The results revealed that incubating the leaf tissues in enzymolysis solution containing 2.0% cellulase onozuka R-10 + 0.5% pectolyase Y-23 + 25.0 mmol/L MES + 0.55 mol/L manitol for 8 h， and purifying them at 1000 rpm centrifuge speed for 6-8 min gave best protoplast with high quality and high activity.