Objective To develop a novel multivariate qualitative analysis method for detecting pathogenic bacteria in food microorganisms based on quantum dot nanoparticles, aiming to meet the current requirements of food safety supervision and the timeliness of short-shelf-life food detection.Methods Quantum dots, with their diverse categories and various synthesis methods, were combined with immunofluorescence analysis to establish a rapid, sensitive, and efficient method for detecting two or more different pathogenic bacteria.Results This method enabled the simultaneous, rapid, sensitive, and efficient detection of foodborne pathogens. When the bacterial concentration was 10³ CFU/mL, pathogens could still be detected within 15 min. Blind sample testing of pathogenic bacteria in dairy products with different substrates showed a 100% agreement rate with traditional methods.Conclusion This method exhibits strong specificity and high sensitivity for pathogen detection, while also being simple to operate and requiring a short detection time, making it highly practical.