Objective To establish ELISA method for H-1 parvovirus, and to apply it in detection. Method Cultured the H-1 parvovirus in rat glioma cell line C6, prepared the viral antigen for coating. Used the purified viral antigen to establish the ELISA method, and compared the ELISA method with the ELISA kit from XpressBio company. Then applied the ELISA method in detection of 35 rat serums. Results The positive serum which be diluted to 1280 can be detected by the ELISA method, there have not cross reaction with positive serum of CPV, MVM and PPV, but there has cross reaction with KRV. 35 pieces of rat serums were detected by the ELISA method, they were all negative, the results were consistent with the kit from XpressBio company. Conclusions The sensitivity and species specificity of the ELISA method for H-1 parvovirus were suitable, the method can be used in detection of H-1 parvovirus in rat serum.