HeNa laser(λ=632.8 nm,P=12 mW) ＆licl was used to irradiate B.subtilis WB3 which had an antagonistic ability on Botrytis cinerea.14 strains that have prominent antagonistic effects were obtained,and the maximum inhibitory bandwidth is 15.7 mm.According to the mortality rates and the size of antagonistic of single mutagenesis group,the optimization condition of irradiation time of 30 min and the density of licl of 1.2% were obtained.After multiplicity mutation,the max inhibiting belt width is L8 that raised more than 14%,however single HeNe laser and licl irradiation could only raise 8.8% and 9%.The strains grew rapidly with stable heredity.The proposed experimental results the HeNa laser ＆ licl mutagenesis of Bacillus subtilistest WB3 show a broad application prospect for the biological control of botrytis cinerea.