We constructed and identified cardiac-specific α-myosin heavy chain (α-MHC) promoter-driven expression vector. α-MHC promoter was amplified by PCR by using mouse genomic DNA as template and inserted into pGEM?-T Easy vector. The inserted fragment was released by enzyme digestion, and then the cytomegalo virus (CMV) promoter in pcDNA3.1(+)-EGFP-hygro vector was replaced by the α-MHC promoter to construct α-MHC-EGFP expression vector. After identification with enzyme digestion, α-MHC-EGFP was transfected into mouse primary cardiomyocytes by electroporation. Green fluorescence could be observed in transfected cardiomyocytes, but not in transfected non-cadiomyocytes. α-MHC-EGFP expression vector was specifically expressed in cardiomyocytes, and could be used to purify embryonic stem cell-derived cardiomyocytes.