The present study aimed to evaluate the role of ascorbic acid in alleviating ammonia-induced muscle quality deterioration and to clarify its regulatory effects on apoptosis, texture, and flavor-related metabolites in rainbow trout (Oncorhynchus mykiss). The results demonstrated that ascorbic acid alleviated ammonia stress-induced inflammatory and apoptotic damage by regulating toll like receptor 5 (TLR5), myeloid differentiation primary response 88 (MyD88), and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) expression, thereby contributing to the restoration of myofibrillar integrity, reduced extracellular gaps, and increased shear force from 14.18 N to 18.26 N (p < 0.05). Ascorbic acid modulated ammonia handling and ion-exchange responses by upregulating glutamine synthetase (GS) expression from approximately 2.3-fold to 6.7-fold and increasing ornithine and citrulline accumulation. Alterations in tricarboxylic acid cycle-related metabolites further suggested that energy metabolism may be involved in the physiological adaptation to ammonia stress. Meanwhile, the ascorbic acid reduced the accumulation of key off-flavor compounds (1-octene-3-alcohol and (E)-2-nonenal), attenuating the earthy–moldy and fishy flavor. This research proposes a potential strategy to improve muscle quality in live transportation.