Programmed cell death (PCD) is a precisely controlled physiological process to sustain tissue homeostasis. Even though the PCD pathways have been explicitly subdivided, the individual cell death process seems to synergistically operate to eliminate cells rather than separately execute signal transduction. Apoptosis is the dominant intracellular PCD subtype, which is intimately regulated and controlled by mitochondria, thus tracing mitochondrial actions could reveal the dynamic changes of apoptosis, which may provide important tools for screening preclinical therapeutic agents. Herein, we exploited an innovative fluorophore Cy496 based on the light-initiated cleavage reaction. Cy496 bears the typical D-π-A structure and serves as a versatile building block for chemosensor construction through flexible side chains. By regulating lipophilicity and basicity through bis-site substitution, we synthesized a series of fluorescence probes and screened a novel mitochondria-targeted ratiometric probe Cy1321, which can real-time evaluate the dynamic changes of mitochondrial micropolarity mediated by bis-cholesterol anchoring. Cy1321 has realized two-color quantification and real-time visualization of polarity fluctuations on chemotherapy agent (cisplatin)-induced apoptosis through flow cytometry and confocal imaging and also achieved the purpose of detecting mitochondria-related apoptosis at the level of tissues. It is envisioned that Cy1321 has sufficient capability as a promising and facile tool for the evaluation of apoptosis and contributing to therapeutic drug screening.