A novel fluorescent probe (BC-PN-2) for the detection of ONOO− was synthesized by conjugating dicyano-vinyl with benzopyran-chromone. The dicyano-vinyl group can be destructed by ONOO− to generate an aldehyde group resulting in strong fluorescence. Compared with the general fluorescence probes, a novel reactive site to detect ONOO− in mitochondria is used in this probe and the results show short response time, high sensitivity and excellent selectivity toward ONOO− among reactive oxygen species (ROS) and reactive nitrogen species (RNS) in PBS. Subsequent fluorescence imaging shows the high membrane permeability, a certain mitochondrial targeting and localization effect of the probe. In addition, the product BC-aldehyde cannot permeate through the membrane. Meanwhile, BC-PN-2 can accurately identify ONOO− in mitochondria by imaging of exogenous ONOO− in HepG2 cells and prove detected ONOO− are from mitochondria.