This study evaluated the impacts of dietary Halimeda opuntia (Chlorophyceae) powder on the growth performance, digestive enzyme activities, immune responses, antioxidant status, gene expression, and intestinal morphology of Nile tilapia (Oreochromis niloticus). A total of 200 fingerlings with an initial average weight of 10.35 ± 0.01 g were randomly allocated into four treatment groups: control, 0.25%, 0.5%, and 1.0% Halimeda powder. Each group was replicated across 5 aquaria, with 10 fish per aquarium. After 8 weeks, the 0.25% Halimeda group (H0.25) showed significant (linear, quadratic, and cubic trends) improvements in growth metrics such as final weight, weight gain, daily weight gain, weight gain percentage, and specific growth rate, compared to the control group. For digestive enzyme activities, the H0.25 group exhibited significantly higher amylase, pepsin, and protease activities, with a cubic response pattern (P ≤ 0.01 or 0.001). Conversely, no significant linear or quadratic effects were observed for triglyceride levels. Meanwhile, the H0.25 group showed significantly higher total protein, albumin, and globulin levels, with cubic trends (P≤ 0.05 or 0.01), and declined liver enzyme activities (aspartate aminotransferase, AST and alanine aminotransferase, ALT) followed quadratic (P ≤ 0.001 for AST, P ≤ 0.01 for ALT) and cubic trends (P ≤ 0.01 for AST, P ≤ 0.05 for ALT). Immune responses, including lysozyme activity, interleukin-1-beta (IL-1β), and tumor necrosis factor alpha (TNF-α) were significantly (linear, quadratic, and cubic trends) influenced by the supplementation, with the most notable effects observed in the H0.25 group. Regarding antioxidant status, glutathione (GSH) and superoxide dismutase (SOD) activities were significantly increased in the H0.25 group, following quadratic and cubic trends (P ≤ 0.05 for GSH, P ≤ 0.001 for SOD). Malondialdehyde (MDA) levels were significantly reduced, showing quadratic and cubic trends (P ≤ 0.001). Gene expression analysis revealed upregulation of both Proliferating Cell Nuclear Antigen (PCNA) and SOD genes in the liver specimens of the 0.25% treated group, with significant linear and quadratic trends (P ≤ 0.01 for both PCNA and SOD). Meanwhile, histological examination indicated enhanced intestinal morphology, with elongated villi and improved absorptive capacity in combination with the dietary administration. In conclusion, a 0.25% inclusion of H. opuntia powder optimizes growth, immune function, antioxidant status, and intestinal health, with significant dose-dependent influences.