Binuclear copper ions at the active site determine the catalysis of tyrosinase (TYR)11TYR: Tyrosinase. l-tyr: l-tyrosine. 4h-Alkaline protease hydrolysate: hydrolysates hydrolyzed by alkaline for 4h. IMAC-Cu: Immobilized Cu ion affinity chromatography. F1: unbound peptides derived from IMAC-Cu. ICP-MS: Inductively coupled plasma mass spectrometry. F2: bound peptides derived from IMAC-Cu. PFRMY: a peptide derived from F2. RGFTGM: a peptide derived from F2. RMSD: root mean square deviations. RMSF: root mean square fluctuations. SASA: solvent accessible surface area. Rg: radius of gyration. MD: molecular dynamics. hpf: hours postfertilization. whose activity can be inhibited by copper’s chelation with other compounds. In this study, tilapia (Oreochromis niloticus) skin was used to generate TYR-inhibitory peptides after being treated by different enzymes and 4 h-Alcaline protease hydrolysate exhibited the highest TYR inhibition and copper chelation. Immobilized metal affinity chromatography was used for purifying copper chelating peptides, among which PFRMY (IC50: 0.43 ± 0.08 mg/mL) and RGFTGM (IC50: 1.61 ± 0.04 mg/mL) exhibited the highest TYR-inhibitory capacity and the lowest docking energy. Both two peptides inhibited TYR in a mixed manner and interacted with key residues binding to copper ions within TYR mainly by hydrogen bonds and hydrophobic forces, while PFRMY had a more compact and stable conjugation with TYR. Zebrafish assay revealed that PFRMY reduced not only melanin synthesis but in vivo TYR activity.