In the present study, the effects of β-glucan administration in water on growth performance and blood status (hematological indexes, immunological and biochemical parameters) were evaluated in Nile tilapia fingerlings (Oreochromis niloticus) under hypoxia-induced stress. In parallel, effect of this prebiotic (beta-glucan) was evaluated on gut microbiota diversity (16S rRNA gene) under normal oxygenation condition. Three treatments were implemented according to beta-glucan water supplementation: 0 (control), 0.1 and 0.3 mg L⁻¹. Blood was collected before hypoxic challenge (BHC) and 9 h after hypoxic challenge (AHC). No significant differences were observed according to growh performance parameters. A significant increase in erythrocyte number and decrease in mean corpuscular volume (P < 0.05) were identified in control group when were under hypoxia. The plasma glucose level was higher in the 0.3 group than in the 0.1 group BHC; however, for all groups, the glucose level increased markedly AHC, and then receded seven days AHC (7AHC, P < 0.05). Total protein, serum lysozyme, plasma lactate, triglyceride, and cholesterol concentrations did not differ between groups. Comparison of the two β-glucan concentrations indicated that the 0.3 mg L⁻¹ concentration promoted an increase in lymphocyte count. The 0.1 group showed the highest survival rate 7 days after hypoxia. Metagenomic analysis revealed a greater number of operational taxonomic units (OTUs) in the 0.1 and 0.3 groups. The Chao richness value was higher in the 0.1 group than in the control group (P <  0.05), with a greater abundance observed for members of the Vibrionaceae family. It is concluded that the concentration of 0.1 mg L⁻¹ was able to increase tolerance to hypoxia, increasing the survival rate and regulating glucose levels. The concentration of 0.3 mg L⁻¹ modulated hematological responses under stress and increased the lymphocyte count before hypoxic challenge.