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Background: Mycoplasma hyopneumoniae is the primary pathogen of mycoplasmal pneumonia of swine (MPS). Vaccination with inactivated bacterin is the most popular and practical measure to control MPS. However, these commercial vaccines have a limited effect on the transmission of microorganism and cannot prevent colonization. Therefore, after immunization with inactivated bacterin, M. hyopneumoniae colonized on the respiratory tract and lungs stimulates the humoral immune responses and produce IgG and IgA antibodies. ELISA is a widely used serological method to detect M. hyopneumoniae antibodies. However, commercial IgG ELISA kit cannot distinguish between inactivated bacterin-induced hyperimmune sera and convalescent sera stimulated by natural infection. SIgA ELISA method is laborious for nasal swab collection, and the amount of each swab sample obtained from the nasal cavity is less compared to the serum sample. Establishment of a discriminative ELISA detecting humoral IgG from convalescent sera but not hyperimmune sera is facilitating to evaluate the natural infection of Mycoplasma hyopneumoniae after inactivated bacterins vaccination. Result: We expressed and purified a recombinant protein named Mhp366-N which contains an epitope recognized by the convalescent sera but not hyperimmune sera. The developed discriminative IgG-ELISA could discriminate between inactivated bacterin-induced hyperimmune sera and convalescent sera, and was reproducible, sensitive, and specific to M. hyopneumoniae antibody produced by natural infection. Compared to SIgA-ELISA method, discriminative IgG-ELISA was more convenient to detect IgG antibody from sera than IgA from nasal swabs, although it is limited sensitivity in the early stages of infection. Additionally, to some extent, it has a potential to avoid the interference of maternally derived IgG antibodies. Conclusions: The established discriminative IgG-ELISA was efficient to judge the serological IgG antibodies induced from natural infection or inactivated vaccine stimulation and provided a useful method to investigate and evaluate the live organism infection after the application of inactivated bacterin.