Conventional culture-based methods for detection of E. coli O157:H7 in foods and water sources are time-consuming, and results can be ambiguous, requiring further confirmation by biochemical testing and PCR. A rapid immunoassay prior to cultivation to identify presumptive positive sample would save considerable time and resources. Immunomagnetic separation (IMS) techniques are routinely used for isolation of E. coli O157:H7 from enriched food and water samples, typically in conjunction with cultural detection followed by biochemical and serological confirmation. In this study, we developed a new method that combines IMS with fluorescence immunoassay, termed immunomagnetic fluorescence assay (IMFA), for the detection of E. coli O157:H7. E. coli O157:H7 cells were first captured by anti-O157 antibody-coated magnetic beads and then recognized by a fluorescent detector antibody, forming an immunosandwich complex. This complex was subsequently dissociated for measurement of fluorescence intensity with Signalyte™-II spectrofluorometer. Experiments were conducted to evaluate both linearity and sensitivity of the assay. Capture efficiencies were greater than 98%, as determined by cultural plating and quantitative real-time PCR, when cell concentrations were <10 5 cells/mL. Capture efficiency decreased at higher cell concentrations, due to the limitation of bead binding capacity. At lower cell concentrations (10–10 4 cells/mL), the fluorescence intensity of dissociated Cy5 solution was highly correlated with E. coli 157:H7 cell concentrations. The detection limit was 10CFU per mL of water. The assay can be completed in less than 3h since enrichment is not required, as compared to existing techniques that typically require a 24h incubation for pre-enrichment, followed by confirmatory tests.