Abstract: This study aimed to purify and characterize protease from Aspergillus oryzae. Ammonium sulfate precipitation,DEAE-FF anion exchange chromatography,Butyl-HP chromatography and Superdux 75 10/300 GL gel filtration chromatography were used to purify the protease from A. oryzae. The molecular weights of the protease was approximately 27 ku. Using casein as a substrate,the Kmwas 1.23g·L-1, and the Vmwas27.03 μg·m L-1·min-1. Moreover,the optimum conditions of the protease were 50 ℃ and p H9.0. The protease had the highest hydrolytic activity to casein while lowest activity to BSA(Bull Serum Albumin). The protease had cleavage ability between-Phe-Val-,-Cys-Gly-,-Glu-Ala and-Arg-Gly-residues in bovine insulin chain B,showing a wide range of residue specificity and high selectivity to hydrophobic amino acids. The results provided a reference for use of proteases from A. oryzae in food industry.