Iron oxide nanoparticles (NPs) were encapsulated with polyaspartamide-based graft copolymers to bind and track the immune cells as imaging probes. Mono-disperse iron oxide NPs with a mean diameter of 10.7 nm were synthesized by the thermal decomposition method, and their shape and distribution were measured by electrophoretic light scattering and transmission electron microscopy. To enhance their biocompatibility, interfacial and hydrodynamic stability, and fluorescence detection, biodegradable polysuccinimide (PSI) grafted with several functional groups of octadecylamine (C18), ethanolamine (EA), ethylenediamine (EDA), 4-(N-maleimidomethyl) cyclohexane carboxylic acid N-hydroxysuccinimide ester (SMCC), and fluorescein isothiocyanate (FITC) was coated on the iron oxide NPs. The structure of the C18/EA/SMCC/FITC-g-PSI copolymer was confirmed using ¹H-NMR and FTIR spectroscopy, and its cell binding ability was investigated by flow cytometry and confocal laser scanning microscopy. The synthesized C18/EA/SMCC/FITC-g-PSI copolymer showed an excellent binding affinity to CD4+ T cells, and was highly biocompatible as the cell viability at the highest polymer concentration of 0.4 mg mL⁻¹ was greater than 85 and 75% after 24 and 48 h, respectively, from MTT assay.