In order to obtain microbial strain suitable for the tenderization of beef, a total of 22 bacteria of producing proteinase were isolated from traditional lobster sauce and wine song in Guizhou. With the screening by using microscopy, analyses of enzyme activities, and TPA value of beef with in treatment of fermented broth, a bacteria defined as D7 was found the optimal strain, and its enzyme activity was 71.68 U/g. The molecular biological identification results showed that it was very similar to Bacillus amyloliquefaciens (JN700123.1), and the similarity was 99%, on the basis of molecular biology identification, using 16S rDNA sequences identification and phylogenetic analyses. Thus this strain was identified as Bacillus amyloliquefaciens.