Using a combination of molecular docking and in vitro activity evaluation, three antifreeze peptides (i.e., PQGPVGNTGPKG, LSGPTGEAGRE and GGRGPEGPAGAR) were selected from 386 tilapia skin collagen peptides, and their interactions with ice crystals were explored. The thermal hysteresis activities of these peptides were 1.57, 1.82, and 1.22 ℃, respectively. After freeze-thaw cycles, catalase retained 57.0%, 66.7%, and 52.3% of its initial activity in the presence of the three antifreeze peptides, respectively, significantly higher than that of the blank control (32%). Molecular docking revealed that hydrogen bonding was the primary driving force for the stable binding of these antifreeze peptides to ice crystal planes. These findings offer an efficient strategy for converting tilapia processing by-products into high-value commercial antifreeze agents.