We report the preparation and characterization of spherical core-shell structured Fe₃O₄–Au magnetic nanoparticles, modified with two component self-assembled monolayers (SAMs) consisting of 3–mercaptophenylboronic acid (3–MBA) and 1–decanethiol (1–DT). The rapid and room temperature synthesis of magnetic nanoparticles was achieved using the hydroxylamine reduction of HAuCl₄ on the surface of ethylenediaminetetraacetic acid (EDTA)-immobilized iron (magnetite Fe₃O₄) nanoparticles in the presence of an aqueous solution of hexadecyltrimetylammonium bromide (CTAB) as a dispersant. The reduction of gold on the surface of Fe₃O₄ nanoparticles exhibits a uniform, highly stable, and narrow particle size distribution of Fe₃O₄–Au nanoparticles with an average diameter of 9 ± 2 nm. The saturation magnetization value for the resulting nanoparticles was found to be 15 emu/g at 298 K. Subsequent surface modification with SAMs against glucoside moieties on the surface of bacteria provided effective magnetic separation. Comparison of the bacteria capturing efficiency, by means of different molecular recognition agents 3–MBA, 1–DT and the mixed monolayer of 3–MBA and 1–DT was presented. The best capturing efficiency of E. coli was achieved with the mixed monolayer of 3–MBA and 1–DT-modified nanoparticles. Molecular specificity and selectivity were also demonstrated by comparing the surface-enhanced Raman scattering (SERS) spectrum of E. coli-nanoparticle conjugates with bacterial growth media.