We report the facile hydrothermal synthesis and surface functionalization of hyperbranched polyethyleneimine (HPEI)-coated iron oxide nanoparticles (Fe3O4-HPEI NPs) for biomedical applications. In this study, Fe3O4-HPEI NPs were synthesized via a one-pot hydrothermal method in the presence of HPEI. The formed Fe3O4-HPEI NPs with primary amine groups on the surface were able to be further functionalized with polyethylene glycol (PEG), acetic anhydride, and succinic anhydride, respectively. The formed pristine and functionalized Fe3O4-HPEI NPs were characterized via different techniques. We showed that the size of the Fe3O4-HPEI NPs were able to be controlled by varying the mass ratio of Fe(II) salt and HPEI. In addition, the formed Fe3O4-HPEI NPs with different surface functionalities had good water dispersability, colloidal stability, and relatively high R2 relaxivity (130-160 mM-1s-1). Cell viability assay data revealed that the surface PEGylation and acylation of Fe3O4-HPEI NPs rendered them with good biocompatibility in the given concentration range, while the pristine aminated Fe3O4-HPEI NPs started to display slight toxicity at the concentration of 50 g/mL. Importantly, macrophage cellular uptake results demonstrated that both PEGylation and acetylation of Fe3O4-HPEI NPs were able to significantly reduce the non-specific macrophage uptake, likely rendering the particles with prolonged circulation time. With the proven hemocompatibility and rich amine conjugation chemistry, the Fe3O4-HPEI NPs with different surface functionalities may be applied for various biomedical applications, especially for magnetic resonance imaging and therapy.