This experiment was performed to examine the impact of dietary Spirulina platensis phycocyanin (SPC) on the growth performance, antioxidant status, histomorphology of certain organs, immune status with analyzing the immunohistochemical expression of CD3 and CD20, and disease resistance of Nile tilapia, Oreochromis niloticus against A. hydrophila infection. Fish (N = 225, 41.41 ± 0.29 g/fish) were fed basal diets supplemented with five levels of SPC 0, 0.5, 1, 1.5, or 2 g kg − 1 diet (SPC0, SPC0.5, SPC1, SPC1.5, and SPC2, respectively) for 10 weeks. The final body weight, average daily weight gain, total weight gain (TWG), and specific growth rate were quadratically increased in SPC0.5 and SPC1 groups. The total weight gain was increased by 43.85 and 52.63% in SPC0.5 and SPC1 groups, respectively. According to the broken line regression model, the optimal SPC supplementation level was 1.5 g kg-1 based on the TWG and feed conversion ratio (FCR). The protein level of the fish body was quadratically raised in SPC1 and SPC1.5 groups. Linear and quadratic rise in the fat content and decrease in the ash content in a level-dependent way. The SPC supplementation enhanced most intestinal morphometric measures with increased hepato-pancreatic acinar secretory activities and the presence of immune cells infiltration, particularly in the SPC2 group. The lipid profile and liver function tests in the serum were not significantly affected by SPC supplementation. SPC supplementation increased the serum catalase and superoxide dismutase (SOD) activity and the reduced glutathione (GSH) level and decreased malondialdehyde (MDA) level. The immune response indicators (interleukin 10 (IL10), lysozyme activity, complement 3, and IgM serum levels) were improved by SPC supplementation. The percentage of immunostaining reaction against a cluster of differentiation 3 (CD3) and a cluster of differentiation 20 (CD20) in the liver was strongly increased by SPC addition in a dose-dependent manner (P < 0.05). Dietary SPC led to 100% survivability following A. hydrophila challenge compared to 10% survivability in the SPC0 group (P < 0.05). It could be concluded that SPC can be supplemented to O. niloticus diets up to 2 g kg − 1 diet to improve health, antioxidant status, immune response, and disease resistance against A. hydrophila infection. The optimal SPC supplementation level was 1.5 g kg− 1 .