Abstract: In this study, we performed multiple real-time quantitative polymerase chain reaction (mRT-qPCR) method with pretreatment of SDS (sodium dodecyl sulfate) and PMA (propidium monoazide) for the rapid and accurate detection of the food-borne pathogens Salmonella spp. (SA), Listeria monocytogenes (LM), and Shiga toxin-producing Escherichia coli (STE) in dairy products. We designed three corresponding groups of specific primers and TaqMan probes based on the conserved regions of the invA gene from SA, the hly gene from LM, and the stx gene from STE, and developed an appropriate mRT-qPCR assay. Furthermore, we also evaluated the effects of PMA pretreatment on eliminating false positive results caused by dead bacterial cells and assessed the specificity, sensitivity, and stability of this assay, as well as its efficacy in detecting bacterially contaminated dairy products. The results revealed that pretreatment with PMA combined with SDS prevented the interference of dead bacterial cells, and we established that the optimal effective PMA concentration was 20 μmol/L. The assay showed high specificity and sensitivity and good stability in detecting the three target strains amplified in the presence of 18 non-target strains, with the lowest limits of detection for the three target strains being up to 10² CFU/mL and the variation coefficients of inter-batch and intra-batch being less than 1%. The results obtained for the detection of causal organisms in bacterially contaminated dairy products using the SDS-PMA-mRT-qPCR assay were comparable to those obtained using the national standard method, with a test cycle of 7 h. These findings indicate that the SDS-PMA-mRT-qPCR assay can be used for the rapid and accurate detection of Salmonella spp., L. monocytogenes, and Shiga toxin-producing E. coli in dairy products and represents an appropriate technique for ensuring the safety of dairy products.