Objective To establish an ELISA assay for the detection of mouse IgG antibody to rabies virus. Methods Orthogonal experiments were used to determine the optimal dilution of the sample and optimal concentration of the secondary antibody. The stability, specificity and sensitivity test of the assay was determined. The assay result of these samples were compared with those of a commercial kit. Results The optimal dilution of the sample was 1∶100, the optimal concentration of the secondary antibody was 40 ng/ mL, and the cutoff value was 0. 121. There was no crossover with the common mouse viruses, and the minimum detection sensitivity was 129 μg/ mL, and the coefficient variation of the three replicates of the sample was less than 10%. The coincidence rate with the commercial kit was 100%. Conclusions An ELISA assay for the detection of a mouse IgG antibody to rabies virus was successfully established, which can be used for detection and analysis of rabies mouse models.