Protein deterioration caused by ice crystals is an important factors affecting the frozen storage of fish. In this study, antifreeze peptides extracted from hydrolysates of sea cucumber intestinal protein with inhibition of protein denaturation were screened and characterized. The peptide Leu-Pro-Glu-Phe-Thr-Glu-Glu-Glu-Lys (LPEFTEEEK), derived from neutral protease hydrolysates of sea cucumber intestinal protein, was investigated for its potential to enhance the quality of salmon fillets during three freeze-thaw cycles. The results showed that the application of LPEFTEEEK effectively maintained the texture of fish fillets, as well as the oxidative and conformation stability of myofibrillar protein during the freezing process. Additionally, molecular dynamics simulations verified that LPEFTEEEK could bind to ice crystals and inhibit their recrystallization, thus preventing organisms from being damaged by freezing. This suggests that LPEFTEEEK holds significant promise as a novel cryoprotective agent for marine-derived antifreeze peptides.