Abstract: To provide a serological method for detecting antibodies against Mycoplasma suis (M. suis), a blocking ELISA was developed based on a monoclonal antibody (MAb) to M. suisMSG1 protein. The conditions for each step were optimized. The optimal concentration of the coating antigen was 0.5 μg·mL-1; sera samples diluted 1 fold and incubated 1.5 h at 37 ℃; MAbHRP dilution is 10 000 fold and incubated 1 h at 37 ℃. The blocking results of one hundred M. suis negative sera samples were statistically analyzed, and the cutoff of blocking ELISA was determined that the samples presenting a percentage inhibition of ≥ 23.71% were considered positive; samples with a calculated percentage inhibition of ≤ 36.35% were rated negative and those presenting a blocking effect between 23.71% and 36.35% were considered inconclusive. The Blocking ELISA proved to be specific, sensitive and it showed high reproducibility and low variability. This method will be useful in clinical detection and epidemiological study on M. suis.