[Objective] This paper aimed to investigate the optimal fermentation of collagenase-producing conditions from Bacillus cereus R75E and obtain the high purity collagenase by protein separation and purification techniques. [Methods] We optimized the fermentation conditions and fermentation mediums for maximum production of collagenase from Bacillus cereus R75E using single factor experiment and orthogonal test. The collagenase-containing crude enzyme was obtained after centrifuging from the overnight fermentation of Bacillus cereus R75E. Then, the target collagenase was sequentially purified by ammonium sulfate precipitation, Butyl FF hydrophobic chromatography and SuperdexTM 200 gel filtration chromatography. Its purity was detected by SDS-PAGE electrophoresis. [Results] The optimal fermentation conditions and mediums for maximum production of collagenase from Bacillus cereus R75E were as follows: the fermentation temperature was 41 °C, the inoculation volume was 6%, the fermentation time was 36 h, the carbon source was 10 g/L glucose, the nitrogen source was 5 g/L peptone, and the initial pH was 7.0. The total crude enzyme activity was increased by 2.9 times compared with that of the non-optimized control. Finally, the target collagenase was purified with a purity of more than 90%. The purification fold and recovery of the target collagenase were reached to 18.4 and 1.1%, respectively. [Conclusion] We get the optimum collagenase producing conditions from Bacillus cereus R75E and construct a technological process for collagenase purification, which lay a foundation for the development and application of microbial collagenase.