A simple molecular fluorescent probe L has been designed, synthesized and characterized. The probe upon interaction with different metals ions show high selectivity and sensitivity for Ni2+ through fluorescence “turn-off” response in acetonitrile, and the fluorescence could be recovered when chelating agent TPEN is added in this system, viz an “on-off-on” process. Its recognition mechanism has been established by fluorescence spectra, 1H NMR study. Moreover, to further explore its utility in biological system, L is selected as a probe for Ni2+ indicator in live cells and shows sensitivity on Ni2+ intracellular distribution. Fluorescence co-localization studies demonstrate that L is a specific mitochondria-targeted fluorescent probe with excellent cell membrane permeability. Furthermore, a rapidly reversible fluorescence imaging of probe L detecting Ni2+ in the mitochondria is successfully achieved. These facts would make this probe a great potential for detecting Ni2+ in biology.