In present study, we put forward an approach to prepare three-layer core-shell Fe3O4@SiO2@Au magnetic nanocomposites via the combination of self-assembling, seed-mediated growing and multi-step chemical reduction. The Fe3O4@SiO2@Au magnetic nanocomposites were analyzed and characterized by transmission electron microscope (TEM), scanning electronic microscope (SEM), energy dispersive spectrometer analysis (EDS), Fourier transform infrared spectroscopy (FT-IR), vibrating sample magnetometer (VSM), and ultraviolet and visible spectrophotometer (UV-Vis). TEM and SEM characterizations showed that the FeO4@SiO2@Au nanocomposites were obtained successfully with three-layer structures, especially a layer of thin, smooth and continuous gold shell. The average diameter of Fe3O4@SiO2@Au nanocomposites was about 600 nm and an excellent dispersity was observed for the as-prepared nanoparticles. EDS characterizations demonstrated that the nanocomposites contained three elements of the precursors, Fe, Si, and Au. Furthermore, FT-IR showed that the silica and gold shell were coated successfully. UV-Vis and VSM characterizations showed that the Fe3O4@SiO2@Au nanocomposites exhibited good optical and magnetic property, and the saturation magnetization was 25.76 emu/g. In conclusion, the Fe3O4@SiO2@Au magnetic nanocomposites with three-layer core-shell structures were prepared. Furthermore, Fe3O4@SiO2@Au magnetic nanocomposites were modified with streptavidin (SA) successfully, and it was validated that they performed low fluorescence background, suggesting that they should have good applications especially in bioassay based on fluorescence detection through bonding the biotinylated fluorescent probes.