Abstract: Objective: In this paper, a detection method suitable for rapid identification of three pathogenic bacteria from food in the school catering was studied. Method: Based on isothermal multiple self-matching-initiated amplification technology (IMSA), strain-specific primers were designed for Salmonella invA gene, Escherichia coli O157:H7/NM rfbE gene and Listeria monocytogenes prfA gene to test the sensitivity, specificity, shortest enrichment time, and minimum detection limit of bacterial content. Food microbiology inspection according to National Food Safety Standards (GB/T 4789.4-2016, GB/T 4789.30-2016, GB/T 4789.36-2016) were the reference methods, and compared the consistency of the two methods. Result: The results showed that the sensitivity of our method to Salmonella, Escherichia coli O157:H7/NM and Listeria monocytogenes were 2.14×10³, 2.79×10³ and 3.62×10³ CFU/mL respectively, the specificity was 100%, the shortest enrichment time of artificial contamination were 6, 8 and 8 hours respectively. The detection limits of the lowest bacteria content were 2.14, 2.79 and 3.62 CFU/25 g respectively, and the consistency of the results of 74 food samples using IMSA method and the reference method was 100%. Conclusion: IMSA technology of pathogenic bacteria detection in food had the advantage of having high sensitivity, strong specificity and accurate results, which could complete the detection of three pathogenic bacteria in food in a short time and was suitable for the rapid identification of pathogenic bacteria in school catering.