In this study, a fast and sensitive sandwich assay has been developed with the combination of immuno-magnetic separation (IMS) and fluorescence techniques to enumerate Escherichia coli (E. coli). Iron oxide core gold shell (Fe3O4@Au) magnetic nanoparticles were prepared and modified with biotinylated anti-bodies specific to E. coli. Fluorescence labels have been constructed by preparing chitosan coated CdTe quantum dots (CdTe QDs) with a diameter of 3 +/- 1 nm. The amounts of magnetic nanoparticles and CdTe QDs are optimized to get the best sensitivity. The calibration graph fluorescence intensity versus E. coli concentration was linear in the range of 10(2)-10(8) cfu mL(-1) with a coefficient of determination (R-2) of 0.9905. The limit of detection (LOD) and limit of quantification (LOQ) values are calculated as 30 and 100 cfu mL(-1), respectively. Selectivity of the method is examined by applying the same procedure to bacteria, namely Enterobacter aerogenes (E. aerogenes), Enterobacter dissolvens (E. dissolvens), Staphylococcus aureus (S. aureus) and Pseudomonas aeruginosa (P. aeruginosa), and no interference has been observed. The applicability of the developed method has also been examined in spiked urine samples. As a result, it was found that, this novel method is sensitive to target E. coli and a rapid method with a total analysis time less than 120 min. (C) 2016 Elsevier B.V. All rights reserved.