A triphenylamine-pyridine derivative, viz., a fluorescence probe was designed and synthesized. The probe displayed an "on-off-on" fluorescence switch, upon the sequential addition of Cu2+ and ethylenediaminetetraacetic acid (EDTA). The detection mechanism was confirmed by using UV-vis, fluorescence and H-1 NMR titration spectra. Interestingly, the probe can accumulate in mitochondria, and thus detect Cu2+ in real-time there, which indicated that probe L can be used to monitor intracellular mitochondrial Cu2+ levels. Further understanding of the biological functions of Cu2+ in mitochondria has important implications for treating cancer and other diseases.