A one step RT-PCR was developed to detect H5 subtypes of avian influenza virus (AIV). A pair of specific oligonucleotide primers was designed for the detection of AIV H5 subtypes according to 221 hemagglutinin (HA) gene sequences. Sensitivity to detection of allantoic fluid by one step RT-PCR reached 10 thousand dilutions and detection of swab samples reached 8 dilutions. The tissue and swab samples infected with H5 subtypes AIV were simultaneity detected by one step RT-PCR and virus isolation method. The results showed that the sensitivity of the assay was lower 10 to 100-fold than virus isolation method and the assay gave an excellent (100 %) correlation with the conventional virus isolation method. H1~H15 subtypes of avian influenza and other avian diseases were detected by the one step RT-PCR. The results showed the assays were high specific, without cross-reaction to other subtype or other avian diseases. Development of one step RT-PCR will provide effective technical support for the rapid diagnosis and surveillance of molecular epidemiology of H5 subtypes AIV.