Nonalcoholic fatty liver disease (NAFLD) poses a significant global health concern, necessitating precise diagnostic tools and effective treatment strategies. Peroxynitrite (ONOO-), a reactive oxygen species, plays a pivotal role in NAFLD pathogenesis, highlighting its potential as a biomarker for disease diagnosis and therapeutic evaluation. This study reports on the development of a near-infrared (NIR) fluorescent probe, designated DRP-O, for the selective detection of ONOO- with high sensitivity and photostability. DRP-O exhibits rapid response kinetics (within 2 min) and an impressive detection limit of 2.3 nM, enabling real-time monitoring of ONOO- dynamics in living cells. Notably, DRP-O demonstrates excellent photostability under continuous laser irradiation, ensuring reliable long-term monitoring in complex biological systems. We apply DRP-O to visualize endogenous ONOO- in living cells, demonstrating its potential for diagnosing and monitoring NAFLD-related oxidative stress. Furthermore, DRP-O effectively evaluates the efficacy of therapeutic drugs in NAFLD cell models, underscoring its potential utility in drug screening studies. Moreover, we confirm DRP-O to enable selective identification of fatty liver tissues in a mouse model of NAFLD, indicating its potential for the early diagnosis of NAFLD. Collectively, DRP-O represents a valuable tool for studying ONOO- dynamics, evaluating drug efficacy, and diagnosing NAFLD, offering insights into novel therapeutic strategies for this prevalent liver disorder.