In the present paper, a novel difluoroboron dipyrromethene-based (BODIPY) fluorescent probe with a conjugated hydrazone group, named BDBH, was developed for the detection of F− with high selectivity and sensitivity. After addition of tetrabutylammonium fluoride (TBAF) into a BDBH solution, the fluorescence was “turned on”, which displayed an 80-fold increasement of emission intensity. The wavelengths of excitation and emission were 518 nm and 613 nm respectively, which were free from the background interference. The titration of 1H NMR (Nuclear Magnetic Resonance) indicated that the interaction involved hydrogen-bonding formation between the acidic H of –NH–CO group and F− at low concentration and deprotonation of N–H group at increasing concentration of fluoride. The theoretical calculations verified that once F− interacted with BDBH, the planarity and conjugation of BDBH enhanced, which resulted in a strong fluorescence emission at 613 nm. Furthermore, confocal fluorescence images of BDBH-incubated HeLa cells verified its potential application as an “off-on” sensor to monitor F− in living cells.