Escherichia coli O157:H7, Staphylococcus aureus, and Salmonella are food-borne pathogens that cause serious gastrointestinal illness and frequent food safety accidents. This study aimed to develop a practical multiplex polymerase chain reaction (mPCR) technique for the simultaneous detection of these food-borne pathogens in culture broth and artificial food matrix. Pathogen-specific DNA sequences in the rfbE, nuc, and invA genes were used as targets to design primers for the identification of E. coli O157:H7, S. aureus, and Salmonella, respectively. As expected, the method produced species-specific bands of amplified products without any contaminating non-specific bands. The highest species specificity was established with primer concentrations of 0.1, 0.2, and 0.4 μM for E. coli O157:H7, S. aureus, and Salmonella, correspondingly. The detection sensitivity of this assay was 103 CFU/mL in culture broth, and the limit of detection was consistent with singleplex PCR in the food sample. The mPCR assay proposed here is an easy and convenient detection method, which will be valuable for microbial epidemiology and food safety investigations.