Novel, thiol-functionalized, and superparamagnetic, silica composite nanospheres (SH-SSCNs) with diameters smaller than 100 nm are successfully fabricated through the self-assembly of Fe3O4 nanoparticles and polystyrene100-block-poly(acrylic acid)16 and a subsequent sol-gel process. The size and magnetic properties of the SH-SSCNs can be easily tuned by simply varying the initial concentrations of the magnetite nanoparticles in the oil phase. By incorporating fluorescent dye molecules into the silica network, the composite nanospheres can be further fluorescent-functionalized. The toxicity of the SH-SSCNs is evaluated by choosing three typical cell lines (HUVEC, RAW264.7, and A549) as model cells, and no toxic effects are observed. It is also demonstrated that SH-SSCNs can be used as a new class of magnetic resonance imaging (MRI) probes, having a remarkably high spin–spin (T2) relaxivity (r2* = 176.1 mM−1 S−1). The combination of the sub-100-nm particle size, monodispersity in aqueous solution, superparamagnetism, and fluorescent properties of the SH-SSCNs, as well as the non-cytotoxicity in vitro, provides a novel and potential candidate for an earlier MRI diagnostic method of cancer.