Escherichia coli has been frequently reported as a major foodborne bacteria contaminated in raw milk. Therefore, the aim of this study was to explore a quantitative real-time PCR (qPCR) technique combined with sodium dodecyl sulfate (SDS) and propidium monoaziede (PMA) to detect viable E. coli in milk. An internal amplification control (IAC) was also added into this reaction system as an indicator of false-negative results. The inclusivity and exclusivity of the primers were tested using DNA from 7 E. coli and 7 other bacterial strains. The concentrations of SDS and PMA were determined according to plate counts and Cq values of qPCR, respectively. A standard curve was established using series diluted E. coli DNA. The reliability and specificity of this method were further determined by the detection of E. coli in spiked milk. The results showed that the optimal concentrations of SDS and PMA were 100 ppm and 40 μM, respectively. A standard curve with a good linear relationship (R² = 0.9925, E = 105%) was obtained. Compared with conventional PCR and PMA–qPCR, the SDS–PMA–qPCR assay was more specific and sensitive in viable E. coli detection (P < 0.001). Therefore, we evaluated and improved the SDS–PMA–qPCR method for detecting viable E. coli in milk.