Background: E. coli O157:H7 is one of the intestinal pathogens which causes serious lesion in gastrointestinal system. Detection of this bacteria that able to produce toxin and is the major responsible for hospital infection, usually done by culture on sorbitol-MacConkey agar which is time-consuming test. The aim of this study was the preparation a fast and precise method in order to identification of this bacterium by molecular method based on PCR technique. Material and Methods: rfbE and stx2b genes were selected for proprietary identification of E. coli O157:H7 and the amplification of them were performed by PCR following designing specific primers. Sorbitol-MacConkey agar was used to verification of growth ability of selected colonies during PCR. Results: By appearance of the bonds belong to rfbE and stx2B genes on agarose gel, the ability of designed primers in gene detection in samples of E .coli O157:H7 was verified. Colonies which selected during PCR have growth potency on sorbitol-MacConkey agar medium. Conclusion: It was revealed that we can prepare a fast, precise and relative comfortable method for detection of E. coli O157:H7 strain by using PCR technique and specific primers than other available methods.