Abstract: The objective of the present study was to develop a duplex real-time reverse transcription polymerase chain reaction(rRT-PCR)assay for the simultaneous detection of both hemagglutinin(HA)and neuraminidase(NA)genes of H9N2 avian influenza viruses. In order to design primers and probes for the detection of HA and NA genes of avian influenza virus subtype H9N2, current available HA and NA gene sequence of avian influenza virus subtype H9N2 from GenBank were aligned, and two different fluorescein(FAM and JOE)were used to label HA and NA probe, respectively. A one-step duplex Taq Man rRT-PCR assay, which could detect both HA and NA genes of the avian influenza virus subtype H9N2 simultaneously was then established. The amplification curve showed that the duplex rRT-PCR assay had a very good amplification efficiency. The specificity detection showed that the duplex rRT-PCR assay could identify HA and NA gene of the avian influenza virus subtype H9N2 specifically, with no cross reactivity being observed with other influenza virus subtypes or respiratory tract viruses. The sensitivity of the duplex rRT-PCR assay was determined to be 10 RNA copies per reaction for both HA and NA genes. Sixty H9N2-infected mice and sixty environmental specimens were tested and compared with conventional PCR. the sensitivity of the duplex rRT-PCR was 100-fold higher than conventional PCR. The results of the duplex rRT-PCR were completely consistent with those of virus isolation. It was concluded that the duplex rRT-PCR assay established in this study was a highly specific and sensitive method that could be used for the diagnosis of avian influenza virus subtype H9N2.