Mitochondria are an indispensable organelle for energy production and regulation of cellular metabolism. The structural and functional alterations to mitochondria instigate pathological conditions of cancer, and aging-associated and neurodegenerative disorders. The normal functioning of mitochondria is maintained by quality control mechanisms involving dynamic fission, fusion, biogenesis and mitophagy. Under conditions of mitophagy and neurodegenerative diseases, mitochondria are exposed to different acidic environments and high levels of reactive oxygen species (ROS). Therefore stable molecular tools and methods are required to monitor the pathways linked to mitochondrial dysfunction and disease conditions. Herein, we report a far-red fluorescent probe (Mito-TG) with excellent biocompatibility, biostability, photostability, chemical stability and turn on emission for selective targeting of the mitochondrial matrix in different live cells. The probe was successfully employed for monitoring dynamic processes of mitophagy and amyloid beta (Aβ) induced mitochondrial structural changes.