In this study, we demonstrated the simultaneous detection of Escherichia coli and Salmonella enteritidis, by coupling immunomagnetic separation (IMS) with quantum dots (QDs) labeling. QDs having different emission wavelengths were conjugated with anti-E. coli and anti-Salmonella antibodies. QD-antibody conjugates were used to label immunomagnetically separated bacteria and the fluorescence intensities were measured for enumerations of both species. The concentrations of primary antibodies used in IMS, the ratio of QDs to antibodies during the conjugation and the concentration of QD-antibody conjugates used in labeling were optimized to enhance the sensitivity of the assay. After labeling bacteria with QDs, the quenching observed between bead-bacteria complex and QDs was eliminated by separating QDs from the complex using sodium dodecyl sulfate solution. The fluorescence intensities due to the capturing of different concentrations of bacteria were measured and the working ranges were found to be 5 x 10(2) to 5 x 10(5) cfu/mL for E. coli and 4 x 10(2) to 4 x 10(5) cfu/mL for S. enteritidis.