Method: A mRT-PCR assay was developed for rapid detection of H5N1 influenza A virus. The selected primers were used in mRT-PCR for simultaneous detection of fragments specific for H5 (371 bp), N1 (292 bp) and M (154 bp). The assay was optimized and the specificity and sensitivity of the assay were tested. The assay was also tested with 14 avian and human specimens. Results: The results showed that the mRT-PCR assay can specifically detect H5N1 influenza A virus from not only poultry but also humans. The sensitivity of the assay was about 10 viral copies. Conclusion: The highlights of the mRT-PCR assay are its rapidity, costeffectiveness, specificity and sensitivity. Thus, the assay can be applied for diagnostic and large-scale screening at times of H5N1 influenza a virus outbreaks.