Both glutathione (GSH) and viscosity play an important role in mitochondria. They are closely related to various physiological and pathological processes and are important biomarkers in cancer analysis. Herein, we developed the first dual responsive fluorescence probe MGV that can simultaneously detect mitochondrial GSH and viscosity. MGV is smart and shows a selective ratiometric response to GSH at 535/650 nm with a significant increase of green fluorescence. MGV can also show a distinct red fluorescence enhancement at 627 nm as viscosity increases. In addition, MGV has low cytotoxicity and good mitochondrial-targeting ability. With these features, MGV was successfully applied to image mitochondrial GSH at dual fluorescence channels and track mitochondrial viscosity changes on the red fluorescence channel. With MGV, the formation of mitochondrial bleb vesicles was observed with nystatin stimulation, and during the Cisplatin-induced apoptosis, both the level of GSH and the viscosity showed an increase. Finally, based on the dual-response to GSH and viscosity, MGV was successfully used for dual-channel imaging of cancer cells/tumors. Overall, this work provided a smart probe for GSH and viscosity and new insights/methods for apoptosis and tumor imaging.