Use of olive flounder byproducts, which constitute nearly 50 % of the total fish mass, offers a promising strategy for maximizing protein-rich resources.In this study, we optimized a Prozyme 2000P® enzyme-assisted olive flounder byproduct hydrolysate (OFBP) for large-scale production using response surface methodology and evaluated its efficacy in muscle-atrophy models. The final OFBP exhibited 87.5 % protein content, with over 60 % of peptides under 700 Da, promoting high bioavailability and absorption. In vitro, OFBP (300 µg/mL) enhanced de novo protein synthesis in murine C2C12 myotubes by more than two-fold compared with the untreated control (p <
0.05). In a zebrafish model of dexamethasone-induced muscle atrophy, 1 % OFBP supplementation restored up to 80 % of normal activity levels and significantly increased muscle cross-sectional area relative to atrophic controls (p <
0.05). Lipidomic analysis further revealed that dexamethasone-associated elevations in ceramide and sphingomyelin were downregulated by OFBP, indicating a protective effect on sphingolipid metabolism. Taken together, these findings demonstrate that enzymatic hydrolysis of fish byproducts can yield a nutritionally rich ingredient that not only mitigates muscle atrophy through enhanced protein synthesis but also modulates lipid homeostasis. This work highlights OFBP as a sustainable, functional food candidate for supporting muscle health and addressing waste-utilization needs.