A rhodamine-triazole fluorescent probe bearing a coumarin moiety RTC was synthesized using the Cu(I)-catalyzed click reaction. The rhodamine-triazole conjugate was highly selective to Cu2+ among other metal ions, including Ca2+, Co2+, Cu2+, Cd2+, Mg2+, Fe2+, Fe3+, Hg2+, Zn2+, Ni2+, Pd2+ and Pb2+ in physiological conditions. Upon the addition of Cu2+, the colorless RTC solution turned pink and exhibited a significant fluorescence emission centered at 578 nm. The binding of Cu2+ induced a hydrolysis reaction, leading to a release of the coumarin unit from the rhodamine probe, as confirmed by mass spectrometric data. From the fluorescence titration, the detection limit of RTC for Cu2+ was determined to be 21 nM (1.3 ppb). The sensor was responsive to Cu2+ in a wide pH range and successfully applied to monitor Cu2+ in HEK293T cells by confocal fluorescence imaging.