Highly cross-linked and monodispersed polyphosphazene nanoparticles (NPs) exhibiting strong fluorescence were prepared by a remarkably simple one-pot polycondensation of hexachlorocyclotriphosphazene (HCCP) and 4,5-dibromofluorescein (DBF). The morphology and size of the resulting poly(cyclotriphosphazene-co-dibromofluorescein) (PCTPDBF) NPs can be facilely tuned by varying the solvent and reactant concentration. The fluorescent DBF units were isolated and “fastened” in the cross-linked structures, which can well suppress the concentration-quenching effect of small fluorescent probes. Hence, the PCTPDBF NPs can emit bright yellow fluorescence at any concentration. The PCTPDBF NPs could enter HeLa and H9C2 cells and were located in the cytoplasm. Combined with their excellent biocompatibility and improved resistance to photobleaching and protein interference, they can be used as ideal fluorescent agents for cell imaging.