This paper presents a novel method based on an inverse microemulsion system to synthesize monodisperse magnetic mesoporous silica nanoparticles (MMSN) with core-shell structure. In this method, the water-in-oil microemulsion system was prepared using of cyclohexane containing silica precursor as a continuous oil phase, discrete water droplets containing magnetic seeds (Fe3O4 nanoparticles) and urea as an aqueous phase, and cetyltrimethylammonium bromide (CTAB) and 1-butanol as a surfactant and co-surfactant, respectively. Unlike the traditional reverse microemulsion method, the magnetic seeds used in this system were first covered by a self-organized two-layer surfactant including oleic acid and CTAB as a good host for silica formation. Hence, by removing the CTAB template from the silica structure, a mesoporous silica shell remains on the surface of Fe3O4 nanoparticles. The effects of catalyst types (urea and NaOH), TEOS content, and reaction temperature on the morphology and size of the prepared samples were investigated. It was found that by rising the reaction temperature from 70 to 120 °C, the thickness of the silica layer was increased from 3 to 17 nm. Moreover, a thicker silica coating (26 nm) was obtained by increasing the TEOS content. Also, the performance of the prepared nanocomposite for drug delivery applications was investigated using 5-fluorouracil (5-Fu) as a drug model in a physiological medium. The obtained results showed that the prepared magnetic mesoporous silica nanocomposite has great potential for biomedical applications.