Abstract: Objective To establish a real-time quantitative RT-PCR assay to detect influenza C virus and prepare reference RNA of influenza C virus. Methods The sequence of influenza C virus was obtained from GenBank. The specific primers and TaqMan probes targeting conserved regions were designed with Beacon Designer 8 software. The HE, NP, MP and NS gene sequences of the virus were synthesized, then the synthesized genes were cloned into pBluescript Ⅱ SK (+) plasmid respectively. The reference RNA was prepared and the detection limit and specificity of the assay were tested with in vitro transcription of RNA. Results The rapid, reliable real-time quantitative RT-PCR assay for the identification of HE, NP, MP and NS gene of influenza C virus was established and reference RNA was prepared. Conclusion The real time quantitative RT-PCR assay can be used in the routine respiratory virus surveillance and clinical rapid detection of influenza C virus.