The design of ratiometric fluorescence probes with large Stokes shift is greatly significant in addressing the major challenges of detecting and imaging target substances in complex organisms. Such probes with a self-calibration function can minimize the interference of probe concentration and excitation light. In this study, a novel fluorophore of NA-Py with large Stokes shift of 158 nm, was easily obtained in one step. As a proof of concept, based on NA-Py, a fluorescence probe NA-BOH with strong intramolecular charge transfer (ICT) structural characteristics is designed for detecting peroxynitrite (ONOO−). NA-BOH exhibits red emission (627 nm) and a larger Stokes shift of 178 nm. Furthermore, it demonstrats rapid response (within 20 min) and high selectivity towards ONOO−, with ratiometric quantitative response (0–20 μM), and high sensitivity (182 nM). Alongside excellent biocompatibility and remarkable mitochondrial targeting ability, NA-BOH is successfully utilized for the proportional imaging of ONOO− in living cells and zebrafish.