Shiga toxin-producing E. coli (STEC) is an important foodborne pathogen causing diarrhea, hemorrhagic colitis and hemolytic-uremic syndrome in humans. STEC is an implicated in the vast majority of outbreaks, widely via consumption of STEC contaminated beef, as important vehicle of transmission of this organism to human. The E. coli O157:H7 serotype is traditionally identified by serological identification of the somatic antigen (O157) and structural flagella (H7). In this study, the bacteria were identified as STEC serotype O157:H7 with three primer pairs that amplified fragments of secD, rfbE and fliC genes in PCR assays. These primer pairs specifically amplified different sizes of target genes: a 244bp region of the E. coli diagnostic marker gene (secD); a 317bp region of the O157 lipopolysacharide (LPS) gene (rfbE); and a 381bp region of the H7 flagellin gene (fliC). The singleplex, duplex and triplex PCR assay developed in this study have a sensitivity limit at 2.8 x 103, 2.8 x 105 and 2.8 x 107 CFU/ml of E. coli O157:H7, respectively. Sensitivity to detect trace amount of E. coli O157:H7 DNA was reduced as the number of primer used was increased for competing to the same DNA template.