Fe3O4 nanoparticles synthesized via thermal decomposition in the organic phase have attracted tremendous research interest because of their unique morphology, size dispersion, and crystallinity. However, their poor water dispersibility strongly limited their development in biomedical applications. Therefore, a phase-transfer strategy through which hydrophobic nanoparticles with good performance in the aqueous phase can be obtained is an extremely critical issue. Herein, we present a large-scale, facile, highly efficient strategy for the phase transfer of oleic acid-coated Fe3O4 nanoparticles via a reverse-micelle-based oxidative reaction. The reverse micelle system improves the efficiency of the interface oxidative reaction and prevents the aggregation of nanoparticles during the reaction, facilitating the transfer of Fe3O4 nanoparticles from the organic phase to the aqueous phase. The transferred Fe3O4 nanoparticles are used as a T-2 contrast agent to perform magnetic resonance imaging of CNE2 cells (nasopharyngeal carcinoma cell line). In addition, the free carboxyl groups on the surface of transferred nanoparticles can also be programmed to permit the conjugation of other molecules, in turn allowing nanoparticles to be extended in biological targeting or biological recognition applications. Therefore, this strategy offers a promising platform for the large-scale, highly efficient phase transfer of oleic acid-capped nanoparticles and may become a new paradigm to promote the development of diverse nanoparticles for widespread biomedical applications.