Avian influenza H9N2 subtype viruses have had a great impact on Iranian industrial poultry production economy since introduction in the country. To approach Rapid and precise identification of this viruses as control measures in poultry industry, a real time probe base assay was developed to directly detect a specific influenza virus of H9N2 subtype -instead of general detection of Influenza A viruses- which has been endemic over two last decades in the country. An Iranian avian influenza virus strain of A/Iran/chicken/772/1998 H9N2 subtype were selected as reference strain for of primers and probe designing. The high agreement value of 99% indicated that the devolved real time assay for detection of H9 subtype viruses could easily replace the conventional method of virus isolation particularly in investigation of viruses like national surveillance plan. The limit of detection was almost one EID50 which was the least real infectious unit could be detected. So it can be said that this sensitive assay provided a powerful tool to not to miss any significant viral biological activity neither in the host body nor in the environment. A high level of correlation coefficient (R2 = 0.998) also indicated a good correlation between Ct values and viral concentrations. , it can be conclude that the real time RT-PCR could be easily replace virus isolation in detection of H9N2 influenza viruses especially in large monitoring program. The ability in quantifying of the virus concentration extends usage of test in more accurate studies.