A low-cost duplex SYBR Green-based, real-time polymerase chain reaction (PCR) for the simultaneous detection of Listeria monocytogenes and Staphyloccocus aureus in foods was developed following selection of optimal primers. For L. monocytogenes, the set targeting the listeriolysin O gene (hlyA primers) was more specific than the one annealing to the metalloprotease gene (mpl primers). For S. aureus, the nuc primers targeting the thermonuclease gene were highly specific. Simplex SYBR Green-based, real-time PCR methods for the separate detection of L. monocytogenes and S. aureus were performed. Finally, the developed duplex real-time PCR was applied to foods spiked with these microorganisms using a simple enrichment step in buffered peptone water at 37C for 18 h. Melting temperatures were sufficiently different for identification with intra and inter-assay coefficients of variation in melting temperature of 0.08% and 0.20%, respectively. Detection limits were 7 colony-forming unit (cfu)/g in coleslaw for L. monocytogenes and 2 cfu/g in raw minced meat for S. aureus, as confirmed using the commercial kits and plate counting.