Simple Summary Long-term hypersaline stress can induce coagulation disorders and splenomegaly and down-regulate the complement pathway in tilapia, which can increase risk in healthy breeding. As a prebiotic, beta-glucan dietary supplementation can significantly reduce enlarged spleen resulting from hypersaline stress. The hematological aspects of the red blood cell count, hematocrit, red cell distribution width, platelet count, and plateletcrit were also decreased by supplementation with dietary beta-glucan. In the spleen and intestine, beta-glucan intake significantly decreased the high expression of immune-related genes due to hypersaline stress resulting from beta-glucan intake in tilapia. beta-glucan supplementation also significantly increased the abundance of beneficial microbiota such as Lactobacillus, Phycicoccus, and Rikenellaceae in the intestine. In summary, beta-glucan intake can relieve tissue damage and optimize the intestinal microbiota of tilapia in brackish water and improve fish health. Long-term exposure to hyperosmotic environments can induce severe immune damage and increase risk in tilapia breeding. As an effective immunoregulator, beta-glucan has attracted extensive attention in nutritional research and given rise to high expectations of improving health status and alleviating organismal damage in tilapia, Oreochromis niloticus, in brackish water. In this study, an 8-week cultivation experiment was conducted on tilapia fed a basal diet or diets with beta-glucan supplementation in freshwater (control) and brackish water. Growth performance, hematological aspects, immune cytokine expression, and the intestinal microbiota of tilapia were analyzed. The results indicated that supplementation with beta-glucan significantly reduced the enlarged spleen of tilapia resulting from hypersaline stress. Tilapia fed beta-glucan showed significantly-greater decreases in the red blood cell count, hematocrit, red cell distribution width, platelet count, and plateletcrit than those fed the basal diet. beta-glucan significantly decreased the high expression of immune-related genes in the spleen induced by hyperosmotic stress. In the intestine, the high migration inhibitory factor-2 (MIF-2) and IL-1 beta gene expression induced by hypersaline stress was significantly reduced. beta-glucan supplementation also significantly increased the abundance of beneficial microbiota such as Lactobacillus, Phycicoccus, and Rikenellaceae. Therefore, dietary beta-glucan supplementation can significantly reduce spleen enlargement and improve immune function in tilapia in brackish water. beta-glucan intake can also optimize the intestinal microbiota of tilapia in brackish water and improve fish health.