A new and simple synthetic strategy allows the direct introduction of phosphoric acid anchoring group and carboxyl functional group into polyethylene glycol (PEG), leading to strong binding to iron oxide nanoparticle surfaces through bidentate or tridentate Fe–O–P coordination bonds, and providing functionality for further conjugation of targeting ligands. The carboxyl–PEG–phosphoric-acid-stabilized nanoparticles exhibited extremely high stability in various harsh aqueous environments. The in vivo magnetic resonance imaging (MRI) results showed that the nanoparticles exhibited a long blood circulation time and accumulated in lymph nodes, which indicates that the stabilized Fe₃O₄ nanoparticles are highly stable in the blood stream and are of exceptionally small hydrodynamic diameter. A promising, rapid, easy synthetic procedure, and a successful in vivo MRI application demonstrate the potential of these functionalized nanoparticles as ideal candidates for biomedical applications.