Abstract: The optimal conditions such as enzyme concentration、enzymatic hydrolysis time、reaction temperature、mycelium culture way、mycelium age and type of osmotic stabilizer for the lywallzyme hydrolysis of the cultured mycelia of Pleurotus eryngii for protoplast production were investigated by single factor experiment. The results showed that the highest production rate of the protoplast was emerged under the conditions that 5 days old liquid mycelium in the enzyme solution of 2% lywallzyme digested for 2.5h at temperature of 29℃ with 0.6mol/L mannitol as osmotic pressure stabilizer. This study has laid the foundation of protoplast breeding of Pleurotus eryngii in the future.