Peroxynitrite anion (ONOO^-), one of the reactive nitrogen species (RNS), plays momentous roles in physiological and pathological processes especially in a range of oxidative stress-related diseases. Moreover, abundant ONOO^- is generated in the liver tissues of drug-induced liver injury. We report herein a novel small molecule fluorescent probe KC-ONOO for monitoring ONOO^- based on boronate. The probe displayed high sensitivity and good selectivity towards ONOO^-. A good linear relationship was observed between the fluorescent intensity at 530 nm and the concentration of ONOO^- ranged 0–10 μM with a detection limit of 1.5 × 10⁻⁸ M. Furthermore, our probe was successfully applied for imaging ONOO^- in living cells and drug-damaged liver tissues with low cytotoxicity, demonstrating the probe KC-ONOO has great potential to further elucidate more biological roles of ONOO^-.