Losses caused by foodborne diseases are enormous in terms of human life, illness, medical costs, and food product recalls. Rapid detection of multiple bacterial pathogens in foods is extremely important to ensure food safety. The objective of this research was to develop a multiplex immunoassay by integrating magnetic nanobeads (MNBs) for immunoseparation with quantum dots (QDs) as fluorescent labels for rapid, sensitive, and simultaneous detection of three major pathogenic bacteria, Salmonella Typhimurium, Escherichia coli O157:H7, and Listeria monocytogenes, in food products. In this research, both streptavidin-conjugated MNBs (30- and 150-nm diameter) and QDs (530-, 580-, and 620-nm emission wavelength) were separately coated with biotinylated anti-Salmonella, anti-E. coli, and anti-Listeria antibodies. The immuno-MNBs were mixed with a food sample to capture the three target bacteria. After being magnetically separated from the sample, the MNB-cell conjugates were mixed with the immuno-QDs to form the MNB-cell-QD complexes, and unattached QDs were removed. The fluorescence intensity of the MNB-cell-QD complexes was measured at wavelengths of 530, 580, and 620 nm to determine the populations of Salmonella Typhimurium, E. coli O157:H7, and L. monocytogenes, respectively. This multiplex immunoassay simultaneously detected Salmonella Typhimurium, E. coli O157:H7, and L. monocytogenes at levels as low as 20 to 50 CFU/ml in food samples in less than 2 h without enrichment. The change in fluorescence intensity was linearly correlated (R(2) > 0.96) with the logarithmic value of bacterial level in the range of 10 to 10(3) CFU/ml. More than 85% of the three target pathogens could be simultaneously separated from food samples. The multiplex immunoassay could be expanded to detect more target pathogens, depending on the availability of specific antibodies and QDs with different emission wavelengths.