Construction of multifunctional/multimodality nanoparticles for cancer diagnosis and therapy has become an attractive area of investigation. In this report, we designed a multimodality nanoprobe for cell labeling, and can be detectable by both magnetic resonance and near infrared (NIR) fluorescence imaging. Multiple hydrophobic superparamagnetic iron oxide (SPIO) nanocrystals are self-assembled into nanocomposites in water phase with the help of partially alkylated hyperbranched polycation, polyethylenimine (PEI), which already conjugated with the indocyanine dye Cy5.5 and can be used for cell imaging under NIR fluorescence imaging. The amphiphilic PEI/SPIO nanocomposites have a strong T 2 relaxivity. The iron uptake process in MCF-7/Adr displays a time dependent behavior. Confocal laser scanning microscopy reveals that the nanoprobes are internalized into the cytoplasm of MCF-7/Adr after 24 h labeling. Both MR and NIR fluorescence imaging showed strong image contrast against unlabeled cells. Under a clinical MRI scanner, labeled cells in gelatin phantom present much darker images than controlled ones. The T2 relaxation rate of the labeled cells is 98.2 s 1 , significantly higher than that of the control ones of 2.3 s 1 . This study provides an important alternative to label MCF-7/Adr at optimized low dosages with high efficiency, and may be useful to label other biologically important cells and track their behaviors in vivo. Construction of multifunctional / multimodality nanoparticles for cancer diagnosis and therapy has become an attractive area of ​​investigation. In this report, we designed a multimodality nanoprobe for cell labeling, and can be detectable by both magnetic resonance and near infrared (NIR) fluorescence imaging. Multiple hydrophobic superparamagnetic iron oxide (SPIO) nanocrystals are self-assembled into nanocomposites in water phase with the help of partially alkylated hyperbranched polycation, polyethylenimine (PEI), which already conjugated with the indocyanine dye Cy5.5 and can be used for cell imaging under NIR fluorescence imaging. The amphiphilic PEI / SPIO nanocomposites have a strong T 2 relaxivity. The iron uptake process in MCF-7 / Adr displays a time dependent behavior. Confocal laser scanning microscopy that the nanoprobes are internalized into the cytoplasm of MCF-7 / Both Adr and 24 h labeling. Both MR and NIR fluorescence imaging showed strong image contrast against unlabeled Cells. Under a clinical MRI scanner, labeled cells in gelatin phantom present much darker images than controlled ones. The T2 relaxation rate of the labeled cells is 98.2 s 1, significantly higher than that of the control ones of 2.3 s 1. an important alternative to label MCF-7 / Adr at optimized low dosages with high efficiency, and may be useful to label other biologically important cells and track their behaviors in vivo.