As a significant kind of reactive oxygen species (ROS), peroxynitrite (ONOO-) plays an indispensable role in many physiological and pathological processes. This study aimed to synthesize a novel dihydro-benzo[4,5]imidazo[1,2-c]quinazoline-based probe 1 for detecting ONOO-. In 99.5% H2O solution, probe 1 displayed a distinct aggregation-induced ratiometric emission (AIRE), and would selectively respond toward ONOO-via a ratiometric fluorescent signal, along with a short response time (<30 s) and ultra-sensitivity (LOD = 17.6 nM). Moreover, the probe was applied for monitoring the concentration fluctuations of ONOO- in HeLa cells and zebrafish.