Abstract: To get safe digestive enzyme producing probiotics from intestine of healthy Litopenaeus vannamei, 576 bacteria strains were isolated. Those bacteria strains, extracellular protease, amylase, and lipase activities were detected through qualitative assay and quantittive assay. 11bacteria strains with three kinds of digestive enzyme and high capacity of enzyme production were screened. Secondly, susceptibility and hemolysis of the11strains were tested to identify their security. In this study, vegetative cell suspensions of 11strains in equal proportions were added in the rearing water of juvenile shrimp(L. vannamei). Then shrimps were challenged with Vibrio anguillarum. After the challeng test, real-time RT-PCR was employed to determine the relative expression of several immune genes. Through the difference of the relative expression we could detemine the protective effect of these strains on juvenile shrimp. Considering digestive enzyme activity, biological safety and protective effect, 4 strains were selected. Similarity analysis of 16S rDNA sequences indicated that NO.1, 2 and 4 showed 100% similarity to Bacillus sp. PCSAS 2-38, B. cereus strain N419 and B. thuringiensis strain EA26.1, respectively, and NO.9 showed 99% similarity to Rhodobacter capsulatus strain PSB-03. This laid the foundation for the probiotic development.