Abstract: In the tanning industry, enzymatic unhairing process show the risk of collagen damage on the grain. In order to ensure the quality of leather, it is importance to regulation of collagenase activity during enzymatic unhairing process. Type I collagenase was mixed with epigallocatechin gallate (EGCG), and its hydrolytic activity to collagen was measured, in addition, the structural effects of EGCG on type I collagenase were explored by ultraviolet spectroscopy, fluorescence spectroscopy, and molecular docking technology. The results showed that EGCG concentration of 100 μmol/L could significantly inhibit the collagen hydrolysis activity of type I collagenase (86.06±0.14)%. EGCG can catalyze Zn(II) ion coordination with the active center of type I collagenase, and has hydrogen bonds and hydrophobic interactions with amino acid residues in the protease molecule, resulting in the fluorescence quenching of proteases and decreased collagenase activity. This work provides a way for the screening and design of collagenase activity regulators.