[Objective] We studied the immunoreactivity of Mycoplasma synoviae lipoprotein P80 (MS P80) and applied it to detect ELISA antibody.[Methods] Firstly, we performed bioinformatics analysis, prokaryotic expression and purification on MS P80, and analyzed its immunoreactivity with 6 positive sera against different MS isolates and other avian pathogens by Western blotting. Then we used the purified rMS P80 as coating antigen to establish an indirect ELISA assay for detection of MS antibodies, and tested its specificity, sensitivity, reproducibility and coincidence rates compared with US IDEXX ELISA kit.[Results] The MS P80 protein was predicted as a lipoprotein that shared 98%-100% homology between different MS strains and 25% to 34% homology with other species. Western blotting analysis proved that rMS P80 protein had good immunoreactivity and specificity. An indirect ELISA assay based on rMS P80 was established and showed no cross-reaction with positive sera against other pathogens. The intra- and inter-assay variation coefficients were less than 5% and 10%, respectively. When compared with the commercial kit, the established ELISA assay was more sensitive, and the total coincidence rate was 86%.[Conclusion] MS P80 has good immunoreactivity, intraspecies conservation and interspecies specificity. Therefore, it can be used as a target antigen for MS antibody detection.