Spirulina platensis is, a freshwater microalga, broadly used worldwide. It not only stimulates the immune systems of aquatic organisms but also provides a protein-rich diet and commonly used in the manufacture of aquafeeds. This study was planned to evaluate the growth performance, hepato-renal, and immune response biomarkers of Spirulina and Betaine on Nile tilapia (Oreochromis niloticus) and their protective effect against infection with Aeromonas hydrophila. O. niloticus juveniles (20.22 ± 0.86 g) were divided into four groups (n = 10 per replicate). For 8 weeks, the first and second groups (TS&TB) were fed with 0.5% and 0.3% concentrations of Spirulina and Betaine supplemented diets, respectively; the third group (TSB) was fed with a Spirulina and Betaine mixed diet; the fourth group was fed with a basal diet (without supplementation, T0), which served as control. Dietary inclusion of Spirulina and Betaine significantly improved (P ˂ 0.05) the weight gain, final weight, and food conversion ratio, especially in the TS group. The activities of hepatic malonaldehyde were unchanged in TS & TSB groups and the muscular significantly decreased (P ˂ 0.05) in the same groups, while both increased in the TB group; meanwhile, levels of glutathione reductase were significantly upregulated in all treated groups. Serum interleukins, TNF- alpha, and IL-10 levels were also significantly reduced in all treatment groups. A significant protective power against pathogenic Aeromonas infection was evidenced in all treated groups. Findings in this study highlight the reputation of Spirulina and Betaine as immunostimulants and protective agents against A. hydrophila infection in O. niloticus.