Influenza A virus is now considered to be widespread in poultry and has demonstrated the ability to infect humans in Iran. For laboratory diagnosis of these respiratory viruses, it is essential to have rapid methods, able to detect viruses in early stages of the infection in clinical specimens. The real-time reverse-transcription polymerase chain reaction n (rRT-PCR) assay has been established as a standard method for Influenza virus type A diagnosis inpatients. In this study, we evaluated a single-tube rRT-PCR assay, targeting to the highly conserved region of matrix (M) gene for detection of the virus. In this experimental study, after preparation of 100throat mucus samples, respective RNA was extracted from the virus by using viral RNA extraction kit. Two specific primers were synthesized, based on the conserved region of Influenza type AM-gene and a home-brewed one-step SYBR Green based rRT-PCR was developed and evaluated for detection of Influenza type A infection in the viral samples, on the basis of melting curve analysis. The presence of M-gene in RNAs, extracted from 53viralsamples, was confirmed by this single-tube rRT-PCR assay, and after 45 amplification cycles, the melting curve analysis revealed the melting temperature (Tm) of 83.2 ± 0.5°C for various viral samples, quite different from that of primer-dimers and the positive samples showed only a small variation in parameters. This study showed that the developed one-step rRT-PCR assay is the proper molecular method for rapid and accurate diagnosis of Influenza A by detection of M-protein encoding gene.