This study was designed to investigate the effects of enzymatically hydrolyzed poultry by-products (EHPB) on the growth and muscle quality of largemouth bass. Different concentrations of EHPB (0.00, 3.10, 6.20, 9.30, and 12.40%) were added to replace fishmeal (0.00 (control), 8.89 (EHPB1), 17.78 (EHPB2), 26.67 (EHPB3), and 35.56% (EHPB4)), respectively, in dietary supplementation. The results revealed that the growth performance and muscle amino acid and fatty acid remained unaltered in EHPB1 (p > 0.05). EHPB1 showed significant reduction in muscle hardness, gumminess, chewiness, and muscle fiber count and exhibited a significant increase in muscle fiber volume. The decrease in muscle hardness, gumminess, and chewiness means that the muscle can have a more tender texture. The expression of protein metabolism-related genes reached the highest levels in EHPB1 and EHPB2 (p < 0.05). The mRNA levels of s6k and igf-1 in EHPB2 and EHPB1 were significantly lower than those in the control group. Compared to the control group, the expression of muscle production-associated genes paxbp-1 was higher in EHPB1, and myod-1, myf-5, and syndecan-4 were higher in EHPB2. The mRNA levels of muscle atrophy-related genes, in EHPB4 and EHPB2, were significantly lower than those in the control group. Therefore, the EHPB1 group plays a role in promoting the expression of genes related to muscle formation. In summary, replacing 8.89% of fishmeal with EHPB in feed has no effect on growth and may improve back muscle quality in largemouth bass.