This study employed a multiplex real-time quantitative polymerase chain reaction (mRT-qPCR) method incorporating SDS (sodium dodecyl sulfate) and PMA (propidium monoazide) pretreatment to enable rapid and accurate detection of three food-borne pathogens Salmonella spp. (SA), Listeria monocytogenes (LM), and Shiga toxin-producing Escherichia coli (STE) in dairy products. Three sets of specific primers and TaqMan probes were designed targeting conserved regions of the invA gene (SA), hly gene (LM), and stx gene (STE), facilitating the development of an optimized mRT-qPCR assay. Furthermore, PMA pretreatment efficacy was evaluated for eliminating false-positive results caused by dead bacterial cells, and the evaluation of the specificity, detection limit, stability, and detection effect in the simulated dairy samples was conducted in this study. The results revealed that pretreatment with PMA combined with SDS prevented the interference of dead bacterial cells, and 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.