Abstract: The protoplast preparation conditions of Pleurotus nebrodensis ’Zhongnong No.1’ were studied by using Pleurotus nebrodensis ’Zhongnong No.1’ as test material.The a Zhongnong No.1’without treatment was used as CK,and the atmospheric room temperature plasma（ARTP）mutagenesis technique was used for the first time to study its effect on the yield of fruiting bodies after mutagenesis,in order to obtain superior strains with higher yield than CK.The results showed that the optimum preparation conditions of protoplasts of Pleurotus nebrodensis ’Zhongnong No.1’obtained by single factor test were as follows,enzymolysis temperature 28 ℃,enzymolysis time3 hours,lywallzyme concentration 20 mg·mL^-1,osmotic stabilizer 0.6 mmol·L^-1 KCl.The optimal time of ARTP mutagenesis was 60 seconds,and the positive mutation rate was the most stable when the lethal rate was 94%.The preliminary screening results of the plate screening test showed that there were 4 strains with fast growth rate,strong laccase and cellulase production ability.Further,the LBL decolorization test was rescreened,and 4 strains ZN-37,ZN-20,ZN-39 and ZN-19with good seed setting were screened out.By measuring the cultivation yield of Pleurotus nebrodensis,the final high-yield strain was determined to be ZN-19,with a total yield of 966.95 g,which was 37 %more than the total yield of the CK group.