Infectious hematopoietic necrosis disease (IHN) is an acute infectious viral disease that can cause sudden death of salmon. Vaccine immunization is the most effective way to prevent and control the disease. At present, there is no commercial vaccine to prevent the disease in China. The objective of the present study was to prepare an inactivated vaccine against IHN and evaluate its protective immunity in Oncorhynchus mykiss. In this study, infectious hematopoietic necrosis viruses (IHNV) were successively cultured on Epithelioma papulosum cyprinid (EPC) cells with different multiplicity of infection (MOI). The optimal proliferation pattern of IHNV on EPC cells was determined by measuring the titer of IHNV in each passage combined with the virus harvest time. IHNV was inactivated by β-propanolactone (BPL) at different final concentrations at 24 °C, and the inactivity was then verified in vitro and in vivo to determine the optimal inactivation condition. Inactivated IHNV prepared with the optimal inactivation protocol was intraperitoneally injected into O. mykiss [(10±2) g)] with different doses, and the protective effect of the inactivated vaccine was analyzed by detecting relative percent survival (RPS) after challenge, expression levels of immune-related factors and serum neutralizing antibody titers at different time post vaccination. It was shown that different proliferation patterns had some effects on the proliferation of IHNV on EPC. We chose MOI of 0.0001 as the best inoculation dose on EPC cells, and the virus was harvested on the 3rd day post inoculation at 15 °C. The in vivo and in vitro safety tests showed that the best inactivation condition was to inactivate IHNV at 24 °C for 24 h with the final concentration of 3.0 mmol/L BPL. 10 μL per fish was chosen as the optimal immunization dose, and more O. mykiss were immunized. The RPS was 91.37%, 84.28%, 84.15% and 47.5% at 7, 21, 45 and 60 d post immunization (d.p.i), respectively, and significant difference was observed on RPS between 60 d.p.i and other time points. Compared with the negative group, the expression levels of Mx-1 and IFN-γ were significantly up-regulated in spleen and head-kidney at 7, 15 and 30 d.p.i, and reached the maximum at 7 d.p.i (5 folds). The expressions of CD4 and IgM genes were significantly up-regulated in spleen and head-kidney at 15 d.p.i. In the detection of neutralizing antibody titer, the average neutralizing antibody titer in O. mykiss serum was 67.25, 43.40 and 29.78 at 30, 45 and 60 d.p.i respectively, with a decreasing trend and significant differences among different groups. The results indicated that the IHN-BPL inactivated vaccine developed in this study could induce specific and non-specific immune response in O. mykiss, and could provide significant immunoprotection, which will provide references for the development of inactivated vaccines against IHN.