The development of fluorescent methods for the detection of metal ions is of great importance due to their diverse environmental and biological roles. Herein, a rhodamine 6G-based off-on fluorescent probe (L1) with a t-butyl pyrrole moiety as the recognition site was designed and synthesized. Photophysical studies show that L1 exhibits excellent sensitivity and selectivity towards Cu2+ to other metal ions in neutral acetonitrile aqueous media. Mechanism studies suggest that the recognition process may associate with a Cu2+ promoted hydrolysis reaction of L1. Furthermore, L1 has been successfully applied in fluorescence imaging of Cu2+ ion in living cells.