To screen the proteolytic enzymes that can efficiently degrade collagen, molecular docking and molecular dynamics (MD) simulations were employed to virtually screen proteases for hydrolyzing tilapia collagen powder. The degree of hydrolysis (DH), molecular weight distribution, kinetic constants, and molecular interaction mechanisms were subsequently determined. Results revealed that the binding energies for the three proteases exhibiting favorable collagen hydrolysis-alkaline protease, bovine trypsin, and neutral protease-were -7.09, -6.74, and -6.62 kcal•mol-1, respectively. Enzymatic hydrolysis assays demonstrated that the alkaline protease exhibited excellent hydrolytic activity toward tilapia collagen powder under optimal conditions (pH value 9, 50 ℃), achieving a DH of 30.22%. The hydrolysates had an average molecular weight of 548.23 Da, with collagen tripeptides accounting for 51.34% of the total. The Michaelis constant (Km) and maximum reaction velocity (Vmax) were 4.26 mg•mL-1 and 1.16 μg•(min•mL)-1, respectively. Thus, alkaline protease outperformed both the neutral protease and trypsin. MD simulations over 50 ns indicated that the complex 1AH2-GPH (alkaline protease) reached equilibrium after 15 ns, with the root mean square deviation fluctuating around 0.175 Å. This complex maintained an average of four hydrogen bonds and a stable radius of gyration (Rg) of approximately 1.67 nm. In contrast, complexes 1NPC-GPH and 3MFJ-GPH exhibited less stability. These findings indicate that the alkaline protease identified through virtual screening exhibits high affinity and specificity for tilapia-derived protein, providing important reference for collagen tripeptide production.