A type of BODIPY derivatives was designed and synthesized by the N-alkylation reaction of meso-(4-pyridinyl)-substituted BODIPY. The water-solubility of entire molecule was improved to a large extent as a result of the formation of cationic quaternary ammonium salt, while the strong fluorescence inherent to the BODIPY dye fragment is extinguished on alkylation of the pyridine N atom due to the photo-induced electron transfer (PET) process. The N-alkylated BODIPY derivative 4, as a novel water-soluble “Turn-On” fluorescent probe for the discrimination of H 2 O 2 , was constructed by incorporating 4-(Bromomethyl)benzeneboronic acid pinacol ester moiety, which showed highly selective fluorescent response to H 2 O 2 against other interferences of ROS and RNS species under physiological conditions, and the reaction mechanism of boronate oxidation was confirmed by 1 H NMR, mass spectrum and optical spectroscopy analysis. As a biocompatible probe in biological systems, probe 4 was successfully applied for monitoring and imaging of H 2 O 2 both in vitro and in vivo using HepG2/LO2 cells and angelfish.