Abstract: To address the contradiction between the short shelf life of pasteurized milk and the time-consuming traditional methods for detecting pathogenic bacteria, a robust multiple polymerase chain reaction (mPCR) method was developed to detect Cronobacter sakazakii, Escherichia coli, and Salmonella in pasteurized milk. The aim was to conduct an in-depth research on the basis of routine mPCR, and improve the accuracy and stability of the method. Firstly, two genes were selected for each target strain, and two sets of specific primers were designed to establish two mPCR amplification systems for dual detection. Then, the range stability of optimized annealing temperatures was selected without changing the sensitivity of the method. Finally, the proposed method was compared with the national standard methods GB 4789.40-2016, GB 4789.38-2012, and GB 4789.4-2016 to simultaneously detect artificially contaminated samples and evaluate the application performance. The results showed that the first set of mPCR could detect three pathogenic bacteria in pasteurized milk with good accuracy and stability in the range of annealing temperature of 59~59.5 ℃ (temperature difference was 0.5 ℃), and the sensitivity was 10 CFU/mL. The second set of mPCR could detect three pathogenic bacteria in pasteurized milk with good accuracy and stability in the range of annealing temperature of 57.5~58.5 ℃ (temperature difference was 1 ℃), and the sensitivity was 10 CFU/mL. The established robust mPCR method was used to detect 50 artificially contaminated samples of pasteurized milk, and the results were consistent with the national standard method. The detection time was significantly reduced to 4 hours compared to the national standard method (detection time was 62~148 h). This method had practical application value for microbial epidemiological investigation and research, especially for the rapid detection and safety control of pathogenic bacteria in pasteurized milk.