Microbial collagenases are secreted by anaerobic as well as aerobic pathogenic and non-pathogenic microorganisms to utilize collagen as a source of nutrition. The collagenase production from aerobic non-pathogenic strains can increase its application by decreasing the production time and avoiding pathogenicity. Keeping this in view, previously, collagenase had been isolated and partially purified by gel permeation chromatography (Sephadex G-200) from an aerobic non-pathogenetic microorganism, Bacillus altitudinis, in our laboratory. Thus, the present study was undertaken to purify further and characterize the collagenase. For this, collagenase was purified by anion exchange chromatography using a DEAE cellulose column. SDS-PAGE was carried out, where a single band of apparent homogeneity with the molecular mass of ~23 kDa was observed. Finally, the purified collagenase was characterized in terms of its stability at different temperature and pH ranges. It showed stability at temperature ranging from 4 to 45°C and pH ranging from 4 to 10 up to 60 minutes. Keywords: Purification and Characterization; Bacillus altitudinis.