Abstract: The UV -mutagenesis of protoplast was conducted on Lyophyllum decastes strain ZY48 -1. Results showed that optimized conditions for preparation and regeneration of Lyophyllum decaste’s protoplast were cell age 72h, 0.6mol/L sucrose, lywallzyme concentration 1% , glusulase concentration 0.5% , and temperature 30℃ for an enzymolysis duration 2.5h. The protoplast yield and regeneration rate were 5.76×10 5 protoplasts/mg and 7.81%. Obtained a mutants ZY481-1, its polysaccharide yield was up to 643.1961mg/g, which revealed a 31.05% increase compared with the original stain. Results of continued cultivation for 10 generation showed that the mutant was excellent strain with steady properties and high content of polysaccharides.