To construct the method of determining global peptidase activity on gram-positive bacillus,a L_9(3~4) orthogonal test based on single-factor experiments was designed to optimize the condition of gram-positive bacillus disruption.The content of total free amino is the main evaluation index.As results,the optimum conditions were additive volume of lysozyme 200 μL,ultrasonic power 400W,ultrasonic time 4 min,time interval 3s.11 G+ bacillus with high global peptidase were obtained from 153 strains through this method,numbered DF2-1,ZM1-1,ZM2-1,ZM3-1,DF3-1,DF3-2-1,ZMJ-5,ZMJ-2-6,ZMJ-1-1,DFM-4,WCF-2.