In order to isolate and purify the antibacterial protein from fermentation broth of Bacillus amyloliquefaciens ASR-12. Fermentation broth from strain ASR-12 was precipitated with ammonium sulfate and desalted by dialysis to obtain the crude protein. The crude protein was subjected to heat treatment and Q Sepharose Fast Flow column chromatography. The purified protein was further identified by LC-MS and its coding gene was cloned for sequencing. 5 absorption peaks were obtained after heat treatment and column chromatography, and active detection results showed that the peak Ⅳ has a higher antagonistic activity, which displayed a single band with about 50 KDa by SDS-PAGE detection.The results of LC-MS identification showed that the antibacterial protein had the highest homology with M42 family peptidase and the coverage reached 80%. The gene encoding M42 family peptidase was cloned from the genome of strain ASR-12. The sequencing results after BLAST alignment showed that their homology reached 100% comparing with M42 family peptidase encoding gene from bacillus amyloliquefaciens B9601-Y2. M42 family peptidase is the main active component in the antibacterial protein produced by strain ASR-12.