A separation and purification method of the inhibitor gene for the transglutaminase activated protease belongs to the separation and purification technological field of the biological products. The invention uses the hygroscopicus streptomyces as the starting bacteria; the inhibitor gene for the transglutaminase activated protease can be prepared after the submerged fermentation of the liquid; after the deposition of the ethanol and the replacement of the cation for the chromatography and hydrophobic chromatography, the inhibitor gene solution for the transglutaminase activated protease can be made and used in the following protein sequencing and property study. The steps of the method are simple; the purity of the enzyme is high; and the method is a simple and effective separation and purification method of the enzyme.