Peroxynitrite (ONOO−), one of the reactive oxygen and nitrogen species (RONS), is famous for its potent oxidation and nitration capabilities and plays crucial roles in various pathological conditions. Given the similar chemical properties of various RONS (e.g. short half-life time, ultralow content, and mutual interconversion), specific detecting of ONOO− over other RONS remains a challenging mission in the complex physiological environment, primarily ONOO− related pathological processes. To decipher the intricate associations, molecular imaging assisted by activity-based sensing (ABS) fluorescent probes has become the “state-of-art” method due to its superiorities, including susceptible and selective detection results, non-invasive and real-time visualization. This review focuses on the research of ABS fluorescent probes that revealed the vital roles of ONOO− in various diseases, such as inflammations, tumors, liver injuries, and brain diseases. Finally, we discuss the bottlenecks and future developing trends for ABS probes which will boost ABS probes’ applications in chemistry, biology, and pharmacology.