Mitochondria, known as the “cellular powerhouse”, play a variety of fundamental roles in various physiological and pathological processes, whose oxidative damage is usually emerging as a crucial induction factor that not only induces apoptosis but may also contribute to epilepsy and some other related diseases. Therefore, it is of notable significance to construct a high-discrimination method for informing the diagnosis and treatment of related diseases. Employing Cys-triggered conjugate addition cyclization-elimination strategy, we rationally constructed a mitochondria-targeted colorimetric and ratiometric near-infrared (NIR) fluorescent probe, named Mito-Pa, for Cys-triggered dual-channel fluorescence imaging of mitochondrial oxidative damage during apoptosis. Taking advantage of good membrane permeability and low cytotoxicity, probe Mito-Pa was successfully applied to visualize the mitochondria during apoptotic and the fluctuation of endogenous and exogenous Cys, or generated upon lipopolysaccharide-induced oxidative stress, in various living systems, including cancer cells, zebrafish, and mice. Thus, our proposed probe Mito-Pa would provide fresh insight for a better understanding of the ultimate relationship of Cys with mitochondrial oxidative damage, enabling easy access to accurately monitor the occurrence and progress of related diseases.