Biomedical imaging using cell labeling is an important technique to visualize cell dynamics in the body. To label cells, thiol-organosilica nanoparticles (thiol-OS) containing fluorescein (thiol-OS/Flu) and rhodamine B (thiol-OS/Rho) were surface-functionalized with polyethyleneimine (PEI) (OS/Flu-PEI and OS/Rho-PEI) with 4 molecular weights (MWs). We hypothesized PEI structures such as brush, bent brush, bent lie-down, and coiled types on the surface depending on MWs based on dynamic light scattering and thermal gravimetric analyses. The labeling efficacy of OS/Flu-PEIs was dependent on the PEI MW and the cell type. A dual-particle administration study using thiol-OS and OS-PEIs revealed differential endosomal sorting of the particles depending on the surface of the NPs. The endosomes in the labeled cells using OS/Flu-PEI and thiol-OS/Rho revealed various patterns of fluorescence termed barcoded endosomes. The cells labeled with OS-PEI