Abstract: Flavivirus universal primer pairs and nested PCR were developed as an efficient method to detect flavivirus, especially new type flavivirus. Four primers Flav P1-Flav P4 located in conserved region of the NS5 gene were designed after alignment of the flavivirus genome and then nested PCR assay was developed for duck flavivirus. Nested PCR was 1 000 times more sensitive than the conventional PCR and it was only able to amplify the flavivirus with the sensibility of 90 copiesL-1. Phylogenetic analysis showed that duck flavivirus belongs to mosquito-born flavivirus, NTAV group, similar with Tembusu and BYD virus. The clinical samples detection showed that the universal primers and nested PCR were highly specific and sensitive. In addition, the station of evolution of duck flavivirus was clarified.