A systematic series of fluorescence chemosensors (1-3) having one, two and three-metal binding sites based on cyclotriphosphazene derivatives bearing bis-, tetra- and hexakis-2-(pyren-1-yl methylene amino) phenoxy units, respectively, were designed, synthesized, and evaluated for their sensing behaviors toward metal ions using UV/Vis and fluorescence spectroscopies. Upon the addition of Hg2+ in both the absence and presence of competitive metal ions, the chemosensors revealed highly selective and sensitive turn-on emission enhancement based on the combined effects of chelation-enhanced fluorescence (CHEF), C?N isomerization and intramolecular pyrene excimer formation, as well as a color change from yellowish to colorless, which was readily detected by the naked eye. According to the Job plot method, the complexation ratios of chemosensors (1-3) with Hg2+ were found to be 1:1, 1:2 and 1:3 (ligand:metal), respectively, consistent with the proposed number of metal binding sites. Furthermore, the binding modes of chemosensors (1-3) with Hg2+ were supported by H-1 NMR spectroscopy. The increasing complexation ratios from 1:1 to 1:3 for chemosensors (1-3) enabled proportionally decreasing values for the detection limit (LOD) with 0.223 M, 0.114 M and 0.050 M, respectively. The cytotoxicity and fluorescence microscopy experiments also demonstrated that chemosensors (1-3) are non-cytotoxic, and can be used as fluorescence imaging sensors for Hg2+ in living cells.