The combination of mesoporous silica nanoparticles and superparamagnetic nanocrystals to fabricate multifunctional platforms presents great potentials for simultaneous imaging and drug delivery. In this work, we have successfully developed a simple one-step approach to synthesize magnetite-loaded dual-mesoporous silica spheres consisting of large pores in the core and small pores in the shell (Fe₃O₄@DMSSs) by embedding oil-soluble Fe₃O₄ into the large pores of DMSSs, which were prepared by employing polystyrene-b-poly(acrylic acid) (PS-b-PAA) and cetyl trimethyl ammonium bromide (CTAB) as dual-templates. The loading amounts of magnetite can be easily adjusted by varying the initial concentrations of Fe₃O₄ nanoparticles in the oil phase. The in vitro test indicates that Fe₃O₄@DMSSs possesses excellent T₂-weighted magnetic resonance (MR) imaging performance with a maximum T₂ relaxivity (r₂) of 421.5 mM_Fe⁻¹ S⁻¹. Furthermore, a high doxorubicin (DOX) loading capacity (65 wt%) was achieved and the obtained DOX-loaded Fe₃O₄@DMSSs (DOX/Fe₃O₄@DMSSs) exhibits pH-sensitive behaviour with accelerated release of DOX in acidic environment. Confocal laser scanning microscopy observation shows that DOX/Fe₃O₄@DMSSs was able to locate in the cytoplasm of MCF-7 cells and release DOX into the nucleus to kill cancer cells. Therefore, it is anticipated that Fe₃O₄@DMSSs can be promising candidates as both T₂-weighted MR contrast agents and drug delivery carriers in further biomedical applications.