Abstract: In this study, we aimed to investigate the major extracellular proteases in Bacillus velezensis, their enzymatic properties, and their specificity toward enzyme cleavage sites during substrate protein hydrolysis. An extracellular protease produced by B. velezensis SW5 was purified using a three-step protocol: ammonium sulfate precipitation, anion exchange chromatography, and cation exchange chromatography. After purification, the protease exhibited a specific activity of 6629.41 U/mg, with an estimated relative molecular weight of 27.49 kDa. Sequence alignment analyses revealed that the purified protease shared a high homology with subtilisin BPN'; a difference of only one amino acid residue was observed in its N-terminal sequences. Studies on enzymatic properties demonstrated that the extracellular subtilisin in B. velezensis SW5 possessed an optimal catalytic temperature and pH of 50 ℃ and 8.5, respectively. This enzyme maintained good stability at temperatures of <40 ℃ and within the pH range of 7.0~9.0; it retained 74.7% of its relative activity even when put in a 20% NaCl solution for 20 h. Additionally, Ca²⁺ and Mg²⁺ markedly enhanced the activities of the purified proteases. Inhibitor screening confirmed that this protease belonged to the serine protease family. Substrate affinity tests revealed that the purified protease exhibited a high affinity for soy protein isolates, casein, and fish protein. Furthermore, peptide bond cleavage experiments revealed that this protease exhibited broad substrate specificity toward amino acid residues. These characteristics indicate that B. velezensis and its extracellular proteases possess excellent application potential in fields such as food fermentation and enzymatic protein hydrolysis.