AbstractReactive oxygen species (ROSs) are an important class of signaling molecules that play a critical role in regulating physiological processes in the human body. Mitochondria are the primary site of ROSs production, and abnormal concentrations of ROSs can lead to the malignant proliferation of cells, resulting in diseases. Therefore, it is crucial to detect ROSs in mitochondria. Fluorometric methods have gained significant attention from scientists because of their ease of observation, simplicity of operation, and noninvasiveness. Among the fluorescent dyes, boron‐dipyrromethene (BODIPY) stands out for its high quantum yield, large molar extinction coefficient, and excellent chemical stability, making it widely used in fluorescent imaging. The common design principle of fluorescent probe for the detection of ROSs includes photo‐induced electron transfer, intramolecular charge transfer, and fluorescence resonance energy transfer. This paper provides an overview of BODIPY‐based fluorescent probes designed for imaging ROSs in mitochondria, covering the sensing mechanisms, molecular engineering strategies, and recent advancements. Additionally, the review provides insight into the potential clinical applications of mitochondria‐targeting BODIPY probes in disease diagnosis.