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Abstract This study investigated the effects of dietary supplementation with the product Hilyses on growth performance, feed utilization, nutrient composition, hematological parameters, serum biochemistry, immune function, antioxidant status, and digestive enzyme activity in juvenile Nile tilapia (Oreochromis niloticus, initial body weight 4.24 ± 0.01 g). The fish were fed diets supplemented with Hilyses at concentrations of 0, 1, 2, or 3 g/kg for a period of 8 weeks. The results showed that supplementation with Hilyses at levels up to 2 g/kg diet significantly improved final body weight, weight gain, specific growth rate, feed efficiency ratio, protein efficiency ratio, apparent protein utilization, and energy utilization compared to the control diet without Hilyses. Carcass crude protein content and moisture were significantly higher in Hilyses-fed groups, while crude lipid content decreased at the 3 g/kg supplementation level. Hilyses supplementation enhanced various hematological parameters, including increased red blood cell count, total leukocyte count, hemoglobin concentration, hematocrit, and mean corpuscular volume. Serum biochemistry and immune function markers like total protein, albumin, complement component C3, IgM, and IgG were significantly elevated in the 2 and 3 g/kg Hilyses groups. Antioxidant enzyme activities (catalase, glutathione peroxidase, total superoxide dismutase) were enhanced, and lipid peroxidation was reduced, in the 2 g/kg Hilyses group. Digestive enzyme activities, particularly protease and lipase, were also improved with Hilyses supplementation. Histological examination showed reduced lipid deposition in the liver and increased branching of intestinal villi at the 2 g/kg Hilyses level. Overall, these results indicated that dietary Hilyses supplementation at 2 g/kg diet optimizes growth, feed utilization, nutrient composition, hematology, immunity, antioxidant status, and digestive function in juvenile Nile tilapia.