Abstract: Cloning of protease gene apr from protease production strain Bacillus tequilensis C9 which was collected from the soil of turpan in Xinjiang Province, establish the expression system of protease gene. Protease gene was amplified by PCR and bioinformatics software for sequence analysis, construction of pET- 28a prokaryotic expression recombinant plasmid, and the recombinant protein was expressed in E. coli BL21 though IPTG in 37℃and enzyme activity was determined. The gene fragment of protease gene apr was 1098 bp, the nucleotides sequence homology was 98%, compared with Bacillus subtilis aprE (AB734697), encoded a mature protein containing 299 amino acids, which was easy to protein solution, strong hydrophilicity, the protein belonged to the peptidases_S8_S53 superfamily protein family, the molecular mass of fusion protein was 29 kDa, protease activity was 28.4 u/mL. The experiments can lay the theoretical foundation for establish the efficient expression system of protease gene.