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An immunobinding test was developed on nitrocellulose paper with monoclonal antibody to diagnose Mycoplasma bovis in cultural isolates from the genital tract of artificially-infected heifers; polymerase chain reaction was used as the gold standard. The sensitivity of the immunobinding test was 5x103 colony-forming units, and was highly specific since there were no cross-reactions with other strains of Mycoplasma or Acholeplasma. Epidemiological sensitivity was 96.43%, epidemiological specificity 100.00%, positive predictive values 100.00%, and negative predictive values were 100.00%. There were no significant differences in diagnostic validity values between the immunobinding test and the PCR test (P>0.05). Thus, the immunobinding test is a reliable and alternative laboratory diagnostic technique for the M. bovis infection of the reproductive tract in female cattle under experimental conditions; however, it should be field tested in order to entirely evaluate the diagnostic validity of the procedure.