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CRISPR/Cas9 system-mediated multi-copy expression of an alkaline serine protease (AoproS8) from Aspergillus oryzaewas successfully built in Aspergillus niger. Furthermore, AoproS8 was continuously knocked in the glaA, amyA, and aamygene loci in A. nigerto construct multi-copy expression strains. The yield of the AoproS8 3.0 strain was 2.1 times higher than that of the AoproS8 1.0 strain. Then, a high protease activity of 11,023.2 U/mL with a protein concentration of 10.8 mg/mL was obtained through fed-batch fermentation in a 5 L fermenter. This is the first report on the high-level expression of alkaline serine proteases in A. niger. AoproS8 showed optimal activity at pH 9.0 and 40 °C. It was used for the production of xanthine oxidase (XOD)-inhibitory peptides from eight food processing protein by-products. Among them, the duck hemoglobin hydrolysates showed the highest XOD-inhibitory activity with an IC50value of 2.39 mg/mL. Thus, our work provides a useful way for efficient expression of proteases in A. nigerand high-value utilization of protein by-products.