Most of the ONOO- fluorescent probes have restricted applications because of their aggregation-caused quenching (ACQ) effect, long response time and low fluorescence enhancement. Herein, we developed a novel AIEgen fluorescent probe (PE-XY) based on a benzothiazole and quinolin scaffold with high sensitivity and selectivity for imaging of ONOO-. The results indicated that probe PE-XY exhibited fast response towards ONOO- with 2000-fold enhancement of fluorescence intensity ratio in vitro. Moreover, PE-XY exhibited a relatively high sensitivity (limit of detection: 8.58 nM), rapid response (<50 s), high fluorescence quantum yield (δ = 0.81) and excellent selectivity over other analytes towards ONOO-in vitro. Furthermore, PE-XY was successfully applied to detect endogenous ONOO- levels in living HeLa cells, C. elegans and inflammatory mice with low cytotoxicity. Overall, this work provided a novel fast-response and highly selective AIEgen fluorescent probe for real-time monitoring ONOO- fluctuations in living systems.