A novel, highly sensitive “on-off-on” fluorescent probe (E)-N’-((1H-imidazol-2-yl) methylene) -4-(5, 5-difluoro-1, 3, 7, 9-tetramethyl-5H-4λ4,5λ4-dipyrrolo [1, 2-c:2′, 1′-f] [1,3,2] diazaborinin-10-yl) benzohydrazide (BDB) was developed in this study, and its structural and spectral properties were characterized by 13C NMR, 1H NMR, X-ray, ESI-MS, ultraviolet and fluorescence photometry. BDB could specifically and efficiently identify Cu2+ among many other metal ions with a fluorescence quenching efficiency of 92.7% and a recognition limit of 0.65 μM in CH3CN/HEPES (7:3, v/v, pH = 7.23) buffer. The energy difference between BDB and BDB-Cu2+ complex was calculated by DFT. The BDB-Cu2+ complex could continuously identify GSH in the same system and show an obvious fluorescence enhancement response with a recovery efficiency of approximately 91% and a low recognition limit of 0.02 μM, which is significantly lower than the physiological concentration of GSH in living cells. The applicability of BDB is verified by the strip test. BDB can also be used to detect GSH in HeLa cells, which expand the application of fluorescent chemical sensors in biology.