The production of monodispersed magnetic nanoparticles with appropriate surface modification has attracted increasing attention in biomedical applications including drug delivery, separation, and purification of biomolecules from the matrices. In the present study, we report rapid and room temperature reaction synthesis of gold-coated iron nanoparticles in aqueous solution using the borohydride reduction of HAuCl4 under sonication for the first time. The resulting nanoparticles were characterized with transmission electron microscopy (TEM), electron spectroscopy for chemical analysis (ESCA), ultraviolet visible spectroscopy (UV-Vis), and X-ray diffraction (XRD). Surface charges and magnetic properties of the nanoparticles were also examined. The pattern of Fe3O4 nanoparticles is face centered cubic with an average diameter of 9.5 nm and the initial reduction of gold on the surface of Fe3O4 particles exhibits uniform Fe3O4-Au nanoparticles with an average diameter of 12.5 nm. The saturation magnetization values for the uncoated and gold-coated Fe3O4 nanoparticles were found to be 30 and 4.5 emu/g, respectively, at 300 K. The progression of binding events between boronic acid terminated ligand shell and fructose based on the covalent bonding interaction was measured by absorbance spectral changes. Immunomagnetic separation was also performed at different E. coli concentration to evaluate capturing efficiency of resulting nanoparticles. Immunomagnetic separation percentages were varied in a range of 52.1 and 21.9% depend on the initial bacteria counts.