Peroxynitrite (ONOO) is one of the sources of oxidation stress involved in many biological signaling pathways. The role of ONOO being a double-edged sword in biological systems drives the development of effective detection tools. In this work, a boronate-based polymeric fluorescent probe PB-PVA was synthesized and the probe performance was evaluated. The probe exhibits ratiometric sensing of ONOO in a range of 0–6 µM. There is good linear relationship between the probe fluorescence intensity ratio and ONOO concentration. The probe also displays moderate selectivity towards ONOO over other ROS. Moreover, it is water-soluble and possesses good biocompatibility which aids the imaging of ONOO in living cells. These properties could make the probe a promising tool in in vitro study related to ONOO