As a typical pathogen-associated molecular pattern (PAMP), β-1,3-glucan can engage with pattern recognition receptors (PRRs) to initiate an immune response. In this study, we investigated the effects of dietary β-1,3-glucan on growth performance, antioxidant capacity, immune response, intestinal health, and bacterial resistance in juvenile Macrobrachium nipponense. Prawns were fed with five experimental diets containing 0%, 0.05%, 0.1%, 0.2%, and 0.4% β-1,3-glucan for eight weeks. The findings demonstrated that the inclusion of β-1,3-glucan improved weight gain and survival rate in prawns. Prawns fed with β-1,3-glucan exhibited elevated activities of hepatopancreatic ACP (acid phosphatase), AKP (alkaline phosphatase), and SOD (superoxide dismutase), while MDA (malondialdehyde) content was reduced. Expression levels of PRRs related genes including LGBP (lipopolysaccharide and β-1,3-glucan binding protein), lectin, and LBP (lipopolysaccharide-binding protein) were significantly increased in prawns fed with β-1,3-glucan. Intestinal flora analysis revealed suppression of Cyanobacteria abundance at the Phylum level and enhancement in Rhodobacter abundance at the genus level in prawns fed with a 0.2% β-1,3-glucan diet. Furthermore, prawns fed with 0.1%, 0.2%, and 0.4% β-1,3-glucan demonstrated significantly higher survival rates following Aeromonas hydrophila infection. In conclusion, β-1,3-glucan can activate PRRs to improve immune responses in M. nipponese. Within the range of β-1,3-glucan concentrations set in this experiment, it is recommended to add 0.18% of β-1,3-glucan to the diet, taking into account the positive effect of β-1,3-glucan on the survival rate of M. nipponensecu.