A new rhodamine derivative (RhS) with a recognition capability for Cu2+ was synthesized and employed as a reversible fluorescent probe for Cu2+ in drinking water and living cells. The spirolactam ring-opening of probe RhS induced by Cu2+ lead to a highly selective fluorescence “turn-on” response toward Cu2+ over other metal ions with micromolar sensitivity. The 1:1 stoichiometric structure between RhS and Cu2+ were supported by Job's plot, MS and DFT theoretical calculations. The analytical results obtained by UV–vis and fluorescence spectrophotometry show that the linear range and the limit of detection (LOD) of the present probe for Cu2+ are 0.50–20.00 and 0.11μmolL−1, respectively. The present probe was applied to the detection of Cu2+ in drinking water with the recoveries ranging from 100.4 to 101.2%, and the fluorescence imaging for living HeLa cells.