Abstract: The purpose of this study was to separate and purify the keratinase fermented by ，and explore its corresponding enzymatic properties. Keratinase was first extracted from fermentation broth of K. paraultunense by the precipitation of ammonium sulfate, and further purified by the DEAE -Sepharose -FF anion exchange and Superdex-75 Gel filtration chromatography. It was found that the keratinase was purified up to 14.55 times with a recovery of 7.24% , and the molecular weight of the purified keratinase is about 29 KDa identified by SDS -PAGE. Furthermore, the optimal reaction temperature of as prepared keratinase is around 80 ℃ ; the optimal reaction pH of this keratinase is 7.5, and its activity is stable in the pH range of 6.0-9.0. Its kinetic constants of Km and Vm for casein as a substrate are 4.37 mg·mL-1 and 14 556 U·(mg·min) -1 , respectively. Meanwhile its Km and Vm for wool keratin are 15.66 mg·mL-1 and 2610 U·(mg·min) -1 , respectively. In addition, Ca2+ could greatly enhance the activity of keratinase, but its activity was strongly inhibited by phenyl methyl sulfonyl fluoride (PMSF).