Chitosan nanoparticles have exhibited potential antibacterial activity or anticancer activity as their unique character. In this study, we investigated the effect of chitosan nanoparticles protect crayfish Procambarus clarkii against WSSV. Chitosan (from crab shell) nanoparticles were prepared by ultrafine milling. The physicochemical properties of the nanoparticles were determined by particle size measure, zeta potential analysis and scanning electron microscope observation. The total hemocyte count (THC), phenoloxidase (PO) and superoxide dismutase (SOD) activity were measured at days 1, 4, 9 and 12, and the survival rate was also recorded after WSSV challenge. The results showed that chitosan nanoparticles could enhance the survival rate of WSSV-challenged crayfish. And crayfish fed diets supplemented with 10 mg/g chitosan nanoparticles (65% mortality) showed a significantly higher survival rate when compared to the control group (100% mortality). The analysis of immunological parameters revealed that 10 mg/g chitosan nanoparticles showed significantly higher level of prophenoloxidase (proPO), superoxide dismutase (SOD) and total hemocyte count (THC) when compared to the control group. It was found that chitosan nanoparticles could inhibit WSSV replication in crayfish. Our results demonstrated that dietary chitosan nanoparticles effectively improve innate immunity and survival of P. clarkii challenged with WSSV.