This study presented a multiplex, single-tube, realtime polymerase chain reaction (RTi-PCR) approach for detecting Staphylococcus aureus, Vibrio parahaemolyticus, and Salmonella typhimurium, three of the more frequent foodborne pathogenic bacteria typically investigated in a variety of foods. New primer sequences were designed for detection of specific gene fragments in the 23s ribosomal RNA, transmembrane transcription regulator, and replication origin sequences of S. aureus, V. parahaemolyticus, and S. typhimurium. Simultaneous amplifications were performed under the optimized reaction conditions. Melting curve analysis using SYBR Green I RTi-PCR analysis produced characteristic Tm values for each target amplicon, demonstrating specific and efficient amplification of the three fragments. Addition of an internal amplification control did not affect detection sensitivity for the target pathogen. The analysis of frequent foodborne pathogenic bacteria in artificially inoculated food demonstrated analytical sensitivity for direct detection of each pathogen using the Chelex method rather than a commercial DNA extraction kit. The assay was sensitive to $10^3$ colony-forming units (CFU)/reaction. With enrichment (2 or 4 h), each species could be detected at $10^1$ CFU/g. These results provided that RTi-PCR is a rapid and costeffective procedure to detect foodborne pathogens. This assay could become a valuable tool for routine microbiological analysis in the food industry.