Microalgae are one of the most important sources of natural bioactive compounds, especially those revealing antioxidant activity such as β-carotene. Thus, this study was to compare the effects of dietary Vitamin C (L-ascorbic acid) or Vitamin E (αtocopherol) with β-carotene extracted from Arthrospira platensis on Nile tilapia (Oreochromis niloticus) on growth, feed utilization, blood indices, antioxidant activity, non-specific immunological indices and related gene expression. Three hundred and sixty apparent healthy Nile tilapia (5.80 ± 0.286 g) were randomly distributed into four groups. Four isonitrogenous and isoenergetic diets were performed. A control diet was compared against three experimental diets supplemented with Vitamin C (0.5 mg kg −1 diet), Vitamin E (1 g kg −1 diet) or β-carotene (0.5 g kg −1 diet) for 10 weeks.According to the results, there was no significant difference in feed intake (p > 0.05) between experimental diets. Dietary Vitamins C and E and β-carotene significantly (p < 0.05) enhanced the weight gain, final body weight, protein efficiency ratio, specific growth rate and apparent protein utilization in all groups, whereas the best FCR (p = 0.017) and the highest weight gain (p = 0.007) were detected in the β-carotene diet. Fish survival rates differed significantly (p < 0.05) amongst treatments, whilst fish fed a diet supplemented with β-carotene recorded the highest survival rate. The supplemental diet with β-carotene boosted the values of the biochemical and haematological parameters (p < 0.05) compared with the control diet. The activities of catalase, superoxide dismutase (SOD) and total antioxidant capacity (T-AOC) in the liver (p = 0.0560; p = 0.0032; p = 0.0353 respectively) and tilapia muscles were higher in fish fed either β-carotene or Vitamin E (p = 0.0579; p = 0.1494; p = 0.2145 respectively) than other groups. The highest values of SOD, CAT and immune globulin M-2 gene expression (p < 0.05) were found in fish fed a diet enriched with β-carotene.These results suggested that the dietary incorporation of β-carotene had a superior impact on growth performance, haemato-biochemical and immune-oxidative stress biomarkers in addition to the associated gene expression of Oreochromis niloticus.