CRISPR/Cas9 system-mediated multi-copy expression of an alkaline serine protease (S8) from was successfully built in . Furthermore, AoproS8 was continuously knocked in the , , and gene loci in to 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 . 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 IC value of 2.39 mg/mL. Thus, our work provides a useful way for efficient expression of proteases in and high-value utilization of protein by-products.