To establish a rapid, sensitive and specific multiplex PCR method for the simultaneous detection of Staphylococcus aureus, Salmonella spp, and listeria monocytogenes. Three pairs of primers have been designed according to the Staphylococcus aureus nuc gene, Salmonella spp IpaB gene, listeria monocytogenes inlA gene. Orthogonal experimental design was used to determine Multiplex PCR amplification system for Food-borne Bacterial Pathogens of four factors (Taq DNA polymerase, Mg2+, dNTP and primers) from four levels; three DNA fragments of 210bp,280bp and 476bp were amplified. The specificity and the sensitivity of this method was valued. Template was prepared using FTA filter; the three food-borne Bacterial Pathogens were simultaneously detected by the multiplex PCR technology which have been designed; The sensitivity of this method was 3.0×102cfu/mL for Staphylococcus aureus, 2.0×102cfu/mL for Salmonella spp, and 3.5×102cfu/mL for listeria monocytogenes. This method lies on its accuracy, rapidity and efficiency in the diagnosis, so it could be a useful method for the simultaneous detection of the three species of bacteria in food.