This study evaluated effects of graded levels of dietary β-glucan (0, 0.5, 1, 2 and 4 g kg−1) on growth performance, haematological parameters, intestinal Vibrio counts, dose requirement and salinity stress resistance in pompano fish Trachinotus ovatus (6.45 g ± 0.06 (SEM)). After 8-weeks of diet feeding, growth was significantly higher in fish fed diets with 0.10% β-glucan compared to fish fed control diet (no β-glucan). Survival increased significantly in fish fed 0.05 and 0.10% β-glucan compared to control diet. There were significant increases in red blood cells (in 0.20% β-glucan diet), in total leukocytes (in 0.05–0.20% β-glucan diet), in both lymphocyte and monocyte count in fish fed 0.10%–0.40% β-glucan diet. However, dietary β-glucan did not affect neutrophil, eosinophil and basophil counts. Intestinal Vibrio counts were reduced in fish fed any level of β-glucan compared to control. In addition, dietary β-glucan levels highly correlated with growth, survival, intestinal Vibrio counts and haematological index. Optimal β-glucan levels for maximal growth of fish were predicted to be 0.122% at day 21 (R2 = 98.53%), 0.120% at day 28 (R2 = 78.55%), 0.115% at day 42 (R2 = 62.21%) and 0.090% at day 56 (R2 = 75.18%), showing a decreasing β-glucan requirement with increasing fish size. Furthermore, optimal β-glucan levels for maximal haematological parameters based on lymphocyte count, was estimated to be 0.120% (R2 = 98.53%) at day 56. Also, fish fed 0.05%–0.20% β-glucan showed better resistance against salinity stress. In conclusion, β-glucan supplementation is effective for improving growth, intestinal Vibrio counts and boosted stress resistance of the pompano fish, T. ovatus.