Abstract: The aim of the present study was to develop a multiplex RT-PCR for simultaneous detection of FMDV serotypes O, A and Asia-1. Four specific primers were designed by Primer Premier 5.0 Software targeting VPI gene of FMDV. The annealing temperature and other reaction conditions were optimized. The results showed that the detection limit of the established method was 1 pg/μL; the detection results of CSFV, PPV, PRV, PRRSV, PCV and other related virus by using the developed method was all negative, indicating the good specificity of the method. FMDV serotypes O, A and Asia-1 could be accurately discriminated by the multiplex RT-PCR method developed in this study which was suitable for rapid detection and molecular epidemiological survey of the three serotype of FMDV.