The infection with Salmonella spp., Listeria monocytogenes are the most contamination events in the milk and dairy products. There are many disadvantages of conventional culture-based methods, which are still recognized as the "gold standard" for identifying pathogenic bacteria. Thus, the aim of current study was to develop reliable and rapid method for the detection of bacteria causing foodborne. For the establishment of PMA Real-time PCR method for codetection of Salmonella spp., Listeria monocytogenes, bacteria strains, including Escherichia coli (ATCC 25922), Listeria monocytogenes (ATCC 19115; ATCC 19111), Salmonella enterica (ATCC 19115), Staphylococcus aureus (ATCC 25923), Vibrio parahaemolyticus (ATCC 17802), Shigella flexneri (ATCC 12022), Bacillus aureus (ATCC 11778) were enrolled into the establishment of current protocol. The concentration of PMA, primer concentration, probe concentration, and primer annealing temperature, specificity and sensitivity were evaluated. As the results, we successfully established the protocol of PMA Real-time PCR for co-detection of Salmonella spp. and Listeria monocytogenes on milk product. The concentration of PMA was determined as 50 μM. The sensitivity of established protocol were 10 1 CFU/ml for detection of Salmonella spp., and 10 2 CFU/ml for detection of Listeria monocytogenes. The current PMA Real-time PCR protocol was applied to detect the contamination of twenty local milk samples. No sample was co-contaminated with Salmonella spp. and Listeria monocytogenes. These results were similar to its performed by conventional culture-based methods. In summary, the current established PMA (50 μM) Real-time PCR could be applied for the co-detection of Salmonella spp. and Listeria monocytogenes on milk and dairy food.