Abstract: The aim of this study is to establish a rapid multiplex PCR assay for detection of H5,H9 subtype avian influenza virus(AIV)and duck Tembusu virus(DTMUV).According to the HA gene sequences of H5 and H9AIV and NS5 gene of DTMUV in GenBank,three primer sets specific to these three kinds of viruses were designed,respectively.The multiplex PCR for simultaneous detection of AIV(H5,H9)and DTMUV was established,and was applied to clinical testing for three kinds of viruses.The specificity and sensitivity of the multiplex PCR were tested.The results showed that the assay could specifically amplify the gene fragments of H5AIV(380bp),H9 AIV(732bp),and DTMUV(250bp),and were all negative for detection of other duck viruses.This method showed a sensitive of 533pg·μL-1 for H5 AIV,56pg·μL-1 for H9,and 6.6ng·μL-1 for DTMUV.Additionally,the detection results of 200 clinical samples indicated that this multiplex PCR method proved a a rapid,sensitive and specific tool for clinical samples detection.