Objective To study the conditions of separation and regeneration of Poria cocos protoplast.Methods Separation and regeneration of P.cocos protoplast under different ages of hyphae,enzyme consistency,and enzymolysic time and stabillizer.Results The results showed that the yield of the protoplast was different under different ages of hyphae,enzyme concentration,enzymolysic time,and stabilizer.Conclusion For strain Z_(10-2),its optimal age of hyphae,enzyme concentration,and stabilizer is 72 h,3%,and 0.5 mol/L mannitol,respectively.The strain Z_(10-3) is 56 h,3%,and 0.6 mol/L mannitol.The frequency of regeneration of the two strains is the best and 7.5?10~(-3)(strain Z_(10-2)) and 1.3?10~(-2)(strain Z_(10-2)) separately when the stabilizer is 0.6 mol/L mannitol.