Marine fish processing by-products are rich in protein and represent valuable resources for the production of functional peptides. In this study, defatted proteins from salmon bones, mackerel heads, and yellow croaker roe were enzymatically hydrolyzed and subsequently subjected to γ-glutamylation to investigate changes in their antioxidant and anti-inflammatory activities. Oxygen radical absorbance capacity (ORAC) was used as an activity-oriented index to screen the single-factor reaction conditions. The antioxidant and anti-inflammatory activities of the products were further evaluated using an H?O?-induced oxidative injury model in HepG2 cells and an LPS-induced inflammatory model in RAW264.7 cells. The selected γ-glutamylation conditions for the three hydrolysates were 10% L-glutamine, L-glutaminase additions of 3%, 5%, and 5%, solid-to-liquid ratios of 6%, 20%, and 14%, respectively, and a reaction time of 6 h. Methylene blue staining showed that treatment with the three marine fish by-product hydrolysates and their γ-glutamylated products at concentrations of 10–250 μg·mL?¹ for 24 h did not significantly reduce HepG2 cell viability compared with the untreated control (P > 0.05). In the H?O?-induced oxidative injury model in HepG2 cells, some γ-glutamylated products increased superoxide dismutase (SOD) activity. At 100 μg·mL?¹, the γ-glutamylated products from salmon bones, mackerel heads, and yellow croaker roe reduced malondialdehyde (MDA) levels from 1.82 μmol·μg?¹ protein in the model group to 0.88, 0.88, and 1.18 μmol·μg?¹ protein, respectively. In the LPS-stimulated RAW264.7 cell model, the γ-glutamylated mackerel head hydrolysate reduced nitric oxide (NO) levels from 37.50 to 20.09 μmol·L?¹ at 100 μg·mL?¹ and tumor necrosis factor-α (TNF-α) levels from 196.08 to 121.93 pg·mL?¹ at 50 μg·mL?¹.Overall, γ-glutamylation showed the potential to enhance the antioxidant and anti-inflammatory activities of marine fish by-product hydrolysates, although the effects varied with the raw material and treatment concentration. These findings provide a theoretical basis for the functional utilization of marine fish processing by-products.