Objective To develop a real-time RT-PCR assay(rRT-PCR) for efficient detection of duck hepatitis virus type 1(DHV-I). Method According to the different gene sequences of DHV-I from different provinces download from NCBI and to find the conserved sequences. One pair of the specific primers and one TaqMan probe were designed. Then reaction parameters were optimized to develop a real-time RT-PCR assay(rRT-PCR). Results This developed rRT-PCR assay could detect 20 template copies of RNA, and its sensitivity was higher than that of the conventional RT-PCR. This rRT-PCR assay was found to be specific and able to detect DHV-I, and no positive results were observed when nucleic acid from Muscovy duck parvovirus, goose parvovims, Newcastle disease and avian influenza virus, egg drop syndrome virus, reticuloendotheliosis virus, duck Tembusu virus, poultry intestinal arc virus were used as rRT-PCR templates. The results of this developed rRT-PCR assay used for 100 duck clinical samples showed a positive rate of 92%, indicating that DHV exists in duck group of Jiangsu province in China. Conclusion This rRT-PCR assay can be used as a rapid tool for detection of DHV-I.