Avian influenza virus (AIV) poses a significant threat to the poultry industry and public health. Among the diverse AIV subtypes, H3, H4, and H5 are frequently detected in waterfowl and live poultry markets (LPM). The expeditious and precise identification of these subtypes is imperative in impeding the dissemination of the disease. In this study, we have developed a triplex real-time PCR assay endowed with the capacity to simultaneously discriminate AIV subtypes H3, H4, and H5. This method showcases remarkable specificity, selectively amplifying H3, H4, and H5 AIV subtypes sans any cross-reactivity with other subtypes or common avian pathogens. Furthermore, this method exhibits high sensitivity, with a detection threshold of 2.1 × 102 copies/μL for H3, H4, and H5 AIV subtypes. Additionally, the assay demonstrates reproducibility, as evidenced by intra- and interassay variability, with a coefficient of variation below 1.5%. A total of 338 cloacal swabs were collected from LPM to evaluate the performance of our assay. The obtained results evinced a high level of concordance with the sequencing data. In summary, our study has developed a triplex real-time PCR method that can be employed in laboratory-based testing and surveillance of AIV. This assay holds promise in augmenting our ability to detect and monitor AIV subtypes, thereby facilitating timely interventions and safeguarding both the poultry industry and public health.