Objective To establish a multiplex xMAP assay for detection of infectious disease antibodies in the serum of transgenic mice by liquid chips of mouse MHV, LCM, ECT and HANT proteins. Methods The fluorescent microspheres were conjugated with mouse MHV, LCM, ECT and HANT proteins, and the amount of protein-conjugates and the working concentrations of antibodies and Streptavidin-PE were optimized, respectively. The liquid chips of mouse MHV, LCM, ECT and HANT were prepared and used to detect the antibodies in the serum samples from 56 transgenic mice, together with the negative, positive and control serum, by the multiplex xMAP assay. The results of xMAP assay were verified by traditional ELISA. Results (1) The optimal conjugated amount of mouse MHV, LCM, ECT and HANT protein was 20 μg, 20 μg, 40 μg and 20 μg, respectively. The optimal concentration of their detection antibodies was 2 μg/mL, 2 μg/mL, 2 μg/mL and 4 μg/mL, and the optimal concentration of Streptavidin-PE for all of them was 2 μg/mL. (2) The results of xMAP assay showed that the MFI and index values of the serum samples from the No. 14, No. 23, No. 55 and No. 56 transgenic mice detected with the liquid chip of MHV were higher than those of the control serum, while other MFI and index values were lower than those of the control serum, indicating that the antibodies of MHV were positive in the No. 14, No. 23, No. 55 and No. 56 transgenic mice and negative in the other mice, and the antibodies of LCM, ECT and HANT were negative in all of the mice. (3) The results of ELISA showed that the A450 and index values of MHV in the No.14, No.23, No.55 and No.56 transgenic mice were higher than those of the control serum, while other A450 and index values were lower than those of the control serum, indicating that the antibodies of MHV were positive in the No.14, No.23, No.55 and No.56 transgenic mice and negative in the other mice, and the antibodies of LCM, ECT and HANT were negative in all of the mice. These ELISA results were consistent with the results of the xMAP assay. Conclusions The multiplex xMAP assay established in this study can be used in the detection of infectious disease antibodies in transgenic mice.