Abstract: In order to establish a competitive ELISA antigen detection method for relative efficacy value of Mycoplasma hyopneumoniae inactivated vaccine, the test used the whole Mycoplasma hyopneumoniae cells as the coating antigen and the anti-Mycoplasma hyopneumoniae high immunity serum as the detection antibody, to establish a Mycoplasma hyopneumoniae competitive ELISA antigen detection method; the sensitivity and specificity of the method were verified, and then the relative efficacy value detection method of the inactivated Mycoplasma hyopneumoniae vaccine was established based on the principle of the parallel line method according to the quantitative response verification, and the intra-assay and inter-assay reproducibility of the method were investigated. The results showed that the competitive ELISA reaction system for Mycoplasma hyopneumoniae was established, with the coating antigen concentration 32 μg/mL, the dilution of the positive serum 1∶25 000, the dilution of the negative serum 1∶320, the blocking solution 5% skimmed milk powder, the blocking time 1.5 h, the dilution of the enzyme-labeled secondary antibody 1∶20 000, the action time 1.5 h, and the TMB color development time 15 min at room temperature. The conditions for the establishment of the test were that the positive serum OD₄₅₀ value was 0.7-2.0, and the negative serum OD₄₅₀ value was ＜ 0.3; the linear correlation coefficient of this method was 0.999 5, and the linear range was 10-1 280 μg/mL; there was no cross-reactivity with eight kinds of heterologous proteins such as Porcine parvovirus, Swine influenza vrus, Mycoplasma hyorhinis. Based on the competitive ELISA antigen detection system of Mycoplasma hyopneumoniae, the relative efficacy value detection method of the inactivated vaccine was successfully established. The intra-assay coefficient of variation of the relative efficacy value detection method was ＜5%, and the inter-assay coefficient of variation was less than 10%; the relative efficacy value was positively correlated with animal-immunized challenge protection test results. The results suggested that the competitive ELISA detection method of relative efficacy value of Mycoplasma hyopneumoniae inactivated vaccine established in this experiment could be used to determine the immune efficacy of inactivated Mycoplasma hyopneumoniae vaccine, instead of animal test detection.