A phenoxazinium-based chemosensor (1) bearing di(2-picolyl)amine unit was successfully synthesized. The result shows that it is a red-emitting and full water-soluble chemosensor for the selective detection of Cu2+ in pure water. The fluorescence on-off mechanism was studied by ab initio calculations. To confirm the suitability of 1 for biological applications, it was employed in the fluorescence detection of the intracellular Cu2+ with cultured KB cells. The results indicated that 1 had good membrane permeability and could be useful for the fluorescence microscopic imaging.