The hydroxyl radical (•OH), a member of the reactive oxygen species (ROS) family, is mainly reactive and toxic, promotes the harmful effects of oxidative stress, and ultimately leads to programmed cell death and organ malfunction. In order to assess the function that the hydroxyl radical (•OH) plays in physiological and pathological processes, it is crucial to detect and image the radical with high sensitivity and selectivity in biological systems. Here, a novel dual-emission fluorescence probe was designed based on a coumarin-quinoline scaffold that could detect coumarin-quinoline •OH with high selectivity, sensitivity, fast responses, and good biocompatibility. This probe is mito-targeting with two well-resolved (172 nm) emission peaks and can be used to image endogenous •OH in living cells and zebrafish under complex conditions. Besides, by tracking the level of hydroxyl radical in mitochondria and nuclei, we have revealed the two different pathways of •OH generation that occurs in cell apoptosis induced by β-lapachone (β-Lap) and lipopolysaccharide (LPS) for the first time by fluorescence probe.