A calcium-dependent protein kinase gene (CDPK) was isolated from the Cypripedium macranthum roots using reverse transcription-PCR (RT-PCR) and rapid amplification of cDNA ends (RACE) techniques. The full-length fragment of CmCDPK gene was 2079 bp, with a complete open reading frame of 1491 bp, which encodes for 496 amino acids. CmCDPK was predicted to be a stable hydrophilic protein, possessing a typical and conserved serine/threonine protein kinase domain and a transmembrane structure domain. Secondary structure of CmCDPK is abundant in α-helices and random coils. By phylogenetic tree analysis, CmCDPK were clustered with CDPKs from Orchidaceae, such as Phalaenopsis equestris and Dendrobium catenatum. The fragment of interest was subsequently cloned into the pBI121 vector, which was verified by colony PCR, restriction enzyme digestion and Sanger sequencing. Taken together, this work isolated a CDPK gene from C. macranthum and generated the plant binary expression vector, which might provide the possibility for making transformation in tobacco and further illustrating the biological function of CmCDPK.