Abstract: A strain AL-13 producing extracellular collagenase was identified by morphological, physiological and biochemical characteristics and 16S rDNA sequencing. In order to obtain a safe, efficient and high-yield method for collagenase processing, with the activity of collagenase as an index, single factor experiments was used to optimize the culture conditions of temperature, pH and inoculation quantity for collagenase producing. Response surface methodology combined with single factors were used to optimize the enzyme-producing medium. The results showed that, AL-13 was identified as Brevibacillus laterosporus. The optimal enzyme production conditions were:Culture time 48 h, culture temperature 25℃, initial pH8.0, inoculum 6% (v/v). Optimal enzyme production medium were:Glucose 8.14 g/L, beef extract 11.63 g/L, CaCl₂ 0.17 g/L, K₂HPO₄ 2.08 g/L, NaCl 9.48 g/L. Under the conditions of enzyme production, the predicted value of collagenase activity was 154.89 U/mL. And the obtained reliability enzyme activity was (153.06±3.73) U/mL under the optimized processing. The result was close to the predicted value, and the relative error was 0.04%. The enzyme production conditions of the collagenase-producing bacteria were successfully optimized, and the enzyme activity (153.06 U/mL) optimized for enzyme production was increased by 9.5 times compared with that before optimization (16.14 U/mL).