Multifunctional magnetite@silica-fluorescein isothioxylate-g-poly(dimethyl amino ethyl methacrylate) (Fe3O4@SiO2-FITC-g-PDMAEMA) tri-layer microspheres with magnetism, fluorescence, and pH-responsive PDMAEMA shell were designed and prepared. The functional PDMAEMA brush was grafted from Fe3O4@SiO2-FITC nanoparticles by the surface-induced atom transfer radical polymerization (SI-ATRP) onto the alkylbromide-modified Fe3O4@SiO2-FITC fluorescent templates, which were synthesized by the co-precipitation of Fe(II) and Fe(III) under basic condition together with the subsequently modified Stober sol–gel technique. These nanoparticles were characterized by transmission electron microscopy (TEM), Fourier-transform infrared spectroscopy (FT-IR), thermogravimetric analysis (TGA), fluorescence spectrum, dynamic light scattering (DLS), and zeta potentials.