Innovative nanocomposites of magnetic and fluorescent multifunctional hollow spheres were developed, which combined magnetic resonance imaging (MRI), magnetic targeting, fluorescent imaging (FI) and drug delivery into one system. Fe3O4 hollow spheres were used as templates for the deposition of polyelectrolyte (PE) multilayers and CdTe quantum dots (QDs) via layer-by-layer (LBL) method. The PE multilayers/QDs coated Fe3O4 hollow spheres showed high magnetization and fluorescence, which can be used as magnetic and fluorescent multimodal imaging agent in vivo. The multifunctional nanomaterials of polyelectrolyte multilayers/QDs coated Fe3O4 hollow spheres were used as controlled drug release system, which showed pH-sensitive drug release over a long time. By combining the multiplexing imaging capability, the magnetic and fluorescent nanocomposites are potential candidates for simultaneous disease diagnosis and therapy.