A multiplex PCR (MPCR) assay was developed for the detection of Staphylococcus aureus using selected primers designated on the genus-specific gap gene, species-specific nuc gene and enterotoxin-producing EntC1 gene. The internal regions amplified had a product size of 933 bp, 273 bp and 531 bp, respectively. This MPCR assay had a sensitivity to detect 10 cells of S. aureus and 100 pg of genomic DNA of S. aureus. The MPCR assay was found to be specific for S. aureus, as it yielded negative results with other tested bacterial pathogens such as Salmonella typhimurium, Vibrio cholerae and Escherichia coli. Therefore, this developed MPCR assay could be used for the simultaneous detection of S. aureus enterotoxin C-producing strains from clinical and environmental samples.