Most glycolipid antigens used for serological tests of Mycoplasma pneumoniae are not M. pneumonia-specific, and can cross-react with other microorganism antigens and body tissues, resulting in false positives. It is important to identify M. pneumonia-specific antigen(s) for serological testing and correct diagnosis. Two epitopes, rP1-534 and rP1-513, of P1 adhesin predicted by bioinformatics were successfully expressed and purified, and could be recognized by serum samples from M. pneumoniae-infected patients and His tag antibodies by Western blot. There was no cross-reactivity between the anti-recombinant proteins serum and other respiratory antigens. A total of 400 patients were investigated, their respiratory specimens tested by PCR, and sera tested by a commercial test kit; 56 with positive sera and positive respiratory specimens were designated as standard positive serum and 63 patients were designated as standard negative serum. The purified recombinant proteins were used as a combination of antigens or separately to test the serum. Serological test demonstrated that rP1-513 of the C terminal of P1 adhesin is a new candidate antigen with greater sensitivity and specificity for IgG and IgM serodiagnosis of M. pneumoniae-infected patients. The results confirmed that rP1-513 could be a useful new antigen for the immunodiagnosis of M. pneumoniae infection.