Abstract: The mycelium of Stropharia rugosoannulate was used as the research material.The reaction conditions were optimized by response surface method based on the single factor test with protoplast yield as response value, in order to provide reference for genetic improvement of S.rugosoannulata in the later stage.The results showed that after the optimization of response surface method, the preparation conditions for the protoplasts were obtained as follows, enzymolysis time was 2.5 hours, enzymolysis temperature was 32.2 ℃,and the mycelial age was 3.8 days, the maximum protoplast yield was 35.8×10~6 CFU·mL^-1,Compared with the theoretical yield(35.15×10~6 CFU·mL^-1),the deviation was small.Therefor, the optimum preparation conditions based on response surface method can obtain higher protoplast yield.