Abstract: Tilapia skin, rich in collagen, is an ideal raw material for preparation of bioactive peptides. To provide references for the high-value utilization of tilapia processing by-products and the development of food protein-derived collagenase inhibitors, we prepared tilapia skin collagenase inhibitory peptide (TSCIP) from tilapia skin by enzymatic hydrolysis, and studied the correlation between its collagenase inhibitory activity and metal ion binding activity. The results show that the tilapia skin product hydrolyzed by alkaline protease for 4 h had the highest collagenase inhibition activity, as well as Zn²⁺ and Mg²⁺ binding activity, and it was mainly composed of small molecular peptides (<1 000 D accounting for 85.65%). Ultraviolet (UV), Fourier transform infrared (FTIR) and circular dichroism (CD) analysis show that the secondary structure of TSCIP changed after binding with Zn²⁺, Mg²⁺ and collagenase, and the β-sheet content of TSCIP increased significantly. Zn²⁺ and Mg²⁺ bind to TSCIP mainly through carboxyl oxygen, amino nitrogen atoms and carbonyl groups, while collagenase binds to TSCIP mainly through amino nitrogen atoms and carbonyl groups.