A method combining immunomagnetic separation (IMS) and surface-enhanced Raman scattering (SERS) was developed to enumerate Escherichia coli (E. coli). Gold-coated magnetic spherical nanoparticles were prepared by immobilizing biotin-labeled anti-E. coliantibodies onto avidin-coated magnetic nanoparticles and used in the separation and concentration of the E. colicells. Raman labels have been constructed using rod shaped gold nanoparticles coated with 5,5-dithiobis-(2-nitrobenzoic acid) (DTNB) and subsequently with a molecular recognizer. Then DTNB-labeled gold nanorods were interacted with gold-coated magnetic spherical nanoparticle-antibody-E. coli complex. The capture efficiency and calibration graphs were obtained and examined in different E. coli concentrations (10¹–10⁷ cfu mL⁻¹). The correlation between the concentration of bacteria and SERS signal was found to be linear within the range of 10¹–10⁴ cfu mL⁻¹ (R² = 0.992). The limit of detection (LOD) and limit of quantification (LOQ) values of the developed method were found to be 8 and 24 cfu mL⁻¹, respectively. The selectivity of the developed immunoassay was examined with Enterobacter aerogenes, Enterobacter dissolvens, and Salmonella enteriditis which did not produce any significant response. The ability of the immunoassay to detect E. coli in real water samples was also investigated and the results were compared with the experimental results from plate-counting methods. There was no significant difference between the methods that were compared (p > 0.05). This method is rapid and sensitive to target organisms with a total analysis time of less than 70 min.