The synthesis, solution stability and 64Cu2+ labeling of magnetite nanoparticles (NPs) coated with different macrocycles is reported, together with the stability of the resulting radioisotope-labeled NPs to transchelation by the competing ligand cyclam, and their stability in blood serum. Three macrocycles, 1,4-bis(2-pyridylmethyl)-1,4,7-triazacyclononane (dmptacn), 1,4,8,11-tetraazacyclotetradecane (cyclam) and 1,4,7,10-tetraazacyclododecane (cyclen), and 3-aminopropyltriethoxysilane were used to modify the magnetite NPs. The ligands were covalently linked to the surface of the NPs with high efficiency by reaction of the corresponding 3-(3-(triethoxysiloxy)propoxy)propan-2-ol derivatives with the NPs. According to transmission electron microscopy (TEM), the uncoated magnetite NPs and macrocycle-functionalized congeners have an average diameter of 6 to 7 nm. The NPs form stable colloidal suspensions in 0.05 M aqueous 2-(N-morpholino)ethanesulfonic acid (MES) buffer, which consist of larger aggregates with a mean hydrodynamic size of about 200 nm. The NPs with the appended macrocycles can be efficiently labeled with 64Cu2+ ions and the radioactivity persists in rat plasma for at least 24 h. Challenge experiments with cyclam also indicate that the radiocopper complexes are highly stable, with the dmptacn-functionalized NPs showing the highest resistance to metal ion leakage. Overall, the dmptacn-functionalized iron oxide NPs provide an excellent platform for the development of robust multimodal cancer imaging/therapeutic agents.