Arrays of magnetic nanoparticle iron oxide (Fe3O4) and gold nanoparticles were successfully loaded onto silicate nanosheets of montmorillonite (MMT) to create nano-assemblies--dubbed ‘magnetic Au-nanoclays’, for magnetic separation and surface-enhanced Raman scattering (SERS) sensing. Exfoliated by ion exchange reaction of layered silicate clay, nanoscale silicate platelets (NSP) were achieved by a polyvalent amine salt as an intercalator, and controlling the spacing between the bismuth silicate sheets to optimize the magnetic two-dimensional SERS nanosheets. Transmission electron microscope (TEM) images showed that the diameter of iron oxide nanoparticles is 5-10 nm and Au nanoparticles is about 10-20 nm; and the size of the nanoclays is about 100 nm×100 nm. Due to the hydrophilicity of the magnetic Au-nanoclays, more hydrophilic bacteria such as the gram-positive bacteria (e.g. S. aureus) were easier to be captured and then separated by magnetic field, compared to the more hydrophobic bacteria such as the gram-negative bacteria (e.g. E. coli). Furthermore, Au nanoparticles can produce the surface-enhanced Raman scattering (SERS) effects, facilitating in situ detection of the captured objects based on their molecular vibrational fingerprints. Magnetic Au-nanoclays were employed to magnetically separate biomolecules (e.g., adenine) and bacteria (e.g., E. coli) in water solution and then facilitate their detection by SERS. The potential applications of such novel magnetic Au-nanoclays to the detection of biomolecules and microorganisms as well as environmental pollutants will be discussed.