Encephalomyocarditis virus (EMCV) is one of the major zoonosis pathogens and can cause acute myocarditis in young pigs or reproductive failure in sows. In this study, a TaqMan-based real-time reverse transcription polymerase chain reaction (RT-PCR) assay targeting 3D gene of EMCV was developed and their sensitivities and specificities were investigated. The results indicated that the standard curve had a wide dynamic range (101–106 copies/μL) with a linear correlation (R2) of 0.996 between the cycle threshold (Ct) value and template concentration. The real-time RT-PCR assay is highly sensitive and able to detect 1.4×102 copies/μL of EMCV RNA, as no cross-reaction was observed with other viruses. These data suggested that the real-time RT-PCR assay developed in this study will be suitable for future surveillance and specific diagnosis of EMCV-infection.