This article reports the fabrication of mesoporous Fe3O4 nano/microspheres with a high surface area value (163 m2/g, Brunauer−Emmett−Teller) and demonstrates their use for drug loading, release, and magnetic resonance imaging (MRI). These monodispersed, mesoporous Fe3O4 nano/microspheres with controllable average sizes ranging from 50 to 200 nm were synthesized using a Fe3O4/poly(acrylic acid) hybrid sphere template and subsequent silica shell formation and removal. We found that the SiO2 coating is a crucial step for the successful synthesis of uniform mesoporous Fe3O4 nano/microspheres. The as-synthesized mesoporous Fe3O4 nanospheres show a high magnetic saturation value (Ms = 48.6 emu/g) and could be used as MRI contrast agents (r2 = 36.3 s−1 mM−1). Trypan blue exclusion and MTT assay (see Supporting Information) cytotoxicity analyses of the nanospheres based on HepG2 and MDCK cells showed that the products were biocompatible, with a lower toxicity than lipofectamine (positive control). Hydrophilic ibuprofen and hydrophobic zinc(II) phthalocyanine drug loading into mesoporous Fe3O4 nanospheres and selected release experiments were successfully achieved. The potential use of mesoporous Fe3O4 nanospheres in biomedical applications, in light of the nano/microspheres’ efficient drug loading and release, MRI, and low cytotoxicity, has been demonstrated. It is envisaged that mesoporous Fe3O4 nanospheres can be used as drug carriers and MRI contrast agents for the reticuloendothelial system; they can also be delivered locally, such as via a selective catheter.