Abstract: Abstract: The effect of different enzyme combination, time of enzymolysis, osmtic pressure on protoplast isolation of peanut (Arachis hypogaea L.) were studied. Young leaves and somatic embryiogenic calli of peanut cultivar Huayu20 were used as materials. The results showed that the appropriate enzyme solution was contained 2% cellulase, 0.2% pectinase and 0.7 mol/L mannitol. When cultured for 10 hours in dark, the protoplast yield was 4.86×105 protoplasts, and the activity was 75.6%. After purification, the protoplast was cultured in the modified liquid MS medium containing 1mg/L NAA and 4 mg/L BAP. The first cell division occurred within 2 to 3 days. Then some of the cells divided and developed into colonies. After 5 to 6 weeks, colonies were transferred in the liquid MS medium supplemented with 2 mg/L 2,4-D and 3 mg/L BAP. After 7 to 8 weeks of incubation, protoplast-derived calli up to 1-2 mm in diameter were transferred onto the solid MS medium supplemented with 1 mg/L NAA and 5 mg/L BAP, for callus proliferation. After 3 to 4 weeks of transferring, the calli grew to 7-9 mm in diameter.