A rapid and accurate real-time quantitative polymerase chain reaction (real-time PCR) method with SYBR Green I was established for detecting Escherichia coli O157:H7. A pair of primers were designed to amplify the eae gene. The dissociation curves showed that the amplification product was very specific. The optimal conditions and standard curve were established. The result indicated that real-time PCR was 1000 times more sensitive than ordinary PCR.