Abstract: Objective: Collagenase from Lysinibacillus sphaericus (LsCol1) was heterologously expressed and purified, and its enzymatic property and substrate specificity were determined. It was then used to hydrolyze tilapia skin collagen to prepare antioxidant peptides, with the aim of providing a theoretical basis for the development of a new collagenase resource and its application in the preparation of bioactive peptides. Methods: The amino acid sequence of LsCol1 was analyzed using BLAST and Clustal Omega, and the three-dimensional structure was predicted using AlphaFold 3. The gene sequence of LsCol1 was obtained using chemical synthesis technology, and it was then heterologously expressed in Escherichia coli. LsCol1 was purified using nickel affinity chromatography, and its enzymatic properties and substrate specificity were studied. Finally, the antioxidant peptide was prepared by hydrolyzing tilapia skin collagen with LsCol1. Results: The full length of LsCol1 gene is 3219 bp and encodes 1072 amino acids. The N-terminal of LsCol1 has a catalytic domain, and the C-terminal accessory domain consists of a polycystic kidney disease-like domain and two collagen-binding domains. The molecular weight of purified recombinant collagenase LsCol1 was 120 kDa. The optimal reaction temperature and pH of LsCol1 were 37 ℃ and 7.5, respectively, and it was relatively stable in the range below 30 ℃ and pH7.0~10.0. Ca²⁺ promoted LsCol1 activity. The substrate specificity experiment showed that the optimal substrate for LsCol1 was tilapia skin collagen. The hydrolysate prepared from tilapia skin collagen displayed good antioxidant activity, with 1,1-diphenyl-2-picrylhydrazyl radical, 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) radical, and hydroxyl radical, scavenging rates of 65.9%, 97.6%, and 32.1%, respectively, at a concentration of 4 mg/mL. Conclusion: The collagenase LsCol1 from L. sphaericus had high catalytic capacity and potential application in the preparation of bioactive peptides.