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Two novel water-soluble fluorescence probes T1 and T2 based on 5(6)-carboxyl rhodamine were designed and synthesized using a regioselective reaction. The probes exhibited highly selective and sensitive recognition toward Cu 2+ over other metal ions in acetonitrile/Tris-HCl buffer solution (2:98, v/v; pH 7.4). Detection limits were 0.4 μM for T1 and 4.50 μM for T2 based on fluorescence titration analysis. Furthermore, probe T1 was successfully applied in cell imaging experiments to monitor Cu 2+ in cells.