Fructooligosaccharides (FOS) are widely researched as prebiotics and immunostimulants in humans and terrestrial animals. However, limited researches on immune functions of FOS were reported in crustacean. In the present study, red swamp crayfish Procambarus clarkii were fed with different concentrations of FOS (2.0, 5.0, 8.0, 10.0 g kg−1 diet), and then the immunostimulatory effects of FOS were evaluated by measuring immune parameters and examining immune-related gene expression. PO and SOD activities were significantly increased in groups fed with 8.0 or 10.0 g kg−1 of FOS compared with those in control group. In addition, significant elevations of SOD and phagocytic activities were also observed in group fed with 5.0 g kg−1 of FOS. Among examined immune-related genes, expressions of crustin1, lysozyme, SOD and pro-PO were significantly up-regulated by feeding with 8.0 or 10.0 g kg−1 of dietary FOS. Survival rates of crayfish challenged with Aeromonas hydrophila were significantly improved by feeding with 5.0, 8.0 or 10.0 g kg−1 of dietary FOS for 30 days. Phagocytic activities and bacterial clearance efficiencies were also significantly increased in groups fed with 8.0 or 10.0 g kg−1 of FOS. The results indicated that dietary FOS effectively induced innate immune responses of red swamp crayfish in a dose-dependent way and could be used as potential immunostimulant in crustacean.