Chitosanase enzymes have the ability to break down chitosan to hitosan oligosaccharidewhich exhibits a significant antimicrobial, antitumor activities. In this study, chitosanase enzyme was prepared from Bacillus tequilensis (S9) which was isolated from poultry soil sample containing decomposed chicken feathers. The production medium was optimized and the higher enzyme activity was observed on the 3 rd day. Themaximum chitosanase production of S9 was 4.112 U/ml on shrimp shell media after optimization. The molecular weight of the S9 chitosanase was 40 kDa (crude enzyme), 29 kDa (partially purified). Furthermore, the prepared Chitooligosaccharides hydrolysategel had stronger antibacterial activity against diabetic foot ulcer pathogens and has potential biocompatibility.