Protein denaturation induced by ice crystal formation is the main contributor to the deterioration of fish quality during frozen storage. This study aims to extract hydrolysates capable of inhibiting protein denaturation from sea cucumber ovum protein using three types of proteases (pepsin, trypsin, and neutral protease). The enzymatic hydrolysates of sea cucumber ovum protein are capable of stabilizing protein structures through interactions and preventing protein unfolding and aggregation caused by various factors; thus, they are potential protectors against protein denaturation. This study investigated the effectiveness of various enzymatic hydrolysates of sea cucumber ovum protein in mitigating the quality deterioration and oxidative reactions of mackerel under freeze-thaw cycles. The results show that an enzymatic hydrolysates of sea cucumber ovum, identified as sea cucumber ovum protein neutral protease hydrolysate, efficiently maintained the textural properties and oxidative and conformational stability of mackerel fillets during frozen storage. In addition, sea cucumber ovum protein neutral protease hydrolysate inhibited water migration and microstructural damage to myofibrillar proteins during the freeze-thaw cycles, thereby maintaining overall fish quality. The results of this study indicate that sea cucumber ovum protein neutral protease hydrolysate is a promising candidate for sea cucumber ovum use in marine-based food preservation technologies.