The invention relates to a method for constructing infectious clone of an encephalomyocarditis virus. The method comprises the following steps: extracting the total RNA of the encephalomyocarditis virus; piecewise amplifying the full-length cDNA of the encephalomyocarditis virus by an RT-PCR method, and separately constructing sub-clones; and directionally cloning each fragment of the cDNA sequentially to a low copy plasmid carrier by a double-enzyme cutting method so as to obtain the infectious clone of the encephalomyocarditis virus. The method has small operation difficulty and strong controllability, and can obtain stable infectious clone of the encephalomyocarditis virus, thereby providing an effective tool for the molecular virology research of the encephalomyocarditis virus and the development of vaccines.