Abstract: To monitor the effectiveness of canine rabies vaccination, the indirect ELISA method had been developed to detect the neutralizing antibodies against rabies virus (RV) by using the main antigenic determinant of glycoprotein (RV G3) as coating antigen. The optimum test conditions were as follows: the optimal concentration of RV G3 coating the ELISA plate was 8 mg/L. The optimal concentration of serum samples and HRP-labeled goat anti-canine IgG was 1∶100 and 1∶3 000 respectively. RV G3 had no reaction with positive serum against canine distemper virus (CDV), canine adenovirus (CAV) or canine parvovirus (CPV) by the specificity test. The coefficients of variation of intro-batch and inter-batch duplicability test were fewer than 10%.The sensitivity was 1∶1 280.Compared with America SYNBIOTICS kit, the coincidence rate of indirect ELISA was 95.6%.Therefore, the indirect ELISA has good specificity, repeatability and sensitivity, which can be a good candidate for routine detection of neutralizing antibodies of canine rabies.