The concentration change of overexpressed mitochondrial glutathione (mGSH) in cancer cells can effectively induce cell apoptosis, therefore the development of mGSH -activated turn-on fluorescent probe for cancer cells is extremely beneficial for chemotherapy research. Herein, coumarin Schiff base fluorescent probe N'-((7-(diethylamino)-2-oxo-2H-chromen-3-yl) methylene) picolinohydrazide (NCMP) was prepared, which exhibited selective fluorescence quenching upon the addition of Cu2+. The conjugate NCMP-Cu2+ complex could be used to selectively detect GSH, presenting an “ON-OFF-ON” fluorescence response pattern. The detection limits were 1.98×10-8 M for Cu2+ and 8.20×10-8 M for GSH, respectively. The binding mode of Cu2+ ion with NCMP was ascertained by Job plot, ESI-MS, FT-IR and DFT methods. Probes NCMP-Cu2+ was almost non-toxic, which could be employed for selective fluorescence imaging of GSH in zebrafish and mitochondria of living cells. Consequently, this developed sensor platform has potential application value for future cancer research and biomedical diagnostics.