Fish feed additives are recently encouraged to improve cultured fish health and production. Dietary supplementation with betaine (Bet) and chromium picolinate (Cr-Pic) is known for its high quality for the benefit of aquaculture. A total of 180 healthy and equal-sized O. niloticus were fed on a non-supplemented basal diet, or a basal diet supplemented with Bet (10g/kg dry feed) (Bet group) or Cr-Pic (800 μg/kg dry feed) (Cr-Pic group) or their combination at the same dosing regimens (Bet + Cr-Pic group) for 6 weeks feeding trial. Our findings revealed that dietary supplementation with Bet and/or Cr-Pic enhanced the non-specific immune parameters of O. niloticus in different manners in all supplemented groups compared to the control group. Bet + Cr-Pic group came over the other two groups and recorded the more pronounced highest level of these immune-relevant parameters. In addition, the antioxidative activity of the serum and tissue homogenates of all the supplemented fish was enhanced. The muscular malondialdehyde (MDA) was significantly decreased, with a nominal decrease in the hepatic one; whereas antioxidant enzyme activities (catalase/CAT), reduced glutathione/GSH), glutathione peroxidase/GPx) and superoxide dismutase/ SOD) were all increased significantly in the muscular and hepatic tissues of the supplemented fish groups compared to the control group. The serum cortisol level was significantly decreased in all supplemented groups compared to the control group. Therefore, dietary supplementation with Bet and Cr-Pic combination is a promising feed additive for their additive effects improving fish immune response and antioxidative status and aquaculture sustainability.