Elucidating the internal relationship between diseases and mitochondrial viscosity remains a great challenge due to the lack of the studies on multidisease living animals. So far, demonstrating abnormal mitochondria viscosity in the fatty liver and tumor living mice has not been achieved. To address this critical challenge, the powerful two-photon and mitochondria-targeted fluorescence probe CBI-V was designed herein for viscosity detection with deep red emission. Utilizing the new probe CBI-V, the mitochondrial viscosity alteration in living systems caused by monensin or nystatin has been monitored sensitively and selectively in real time. Moreover, the probe is capable of imaging mitochondrial viscosity in the inflammatory models at the cell, zebrafish, and mice level. Importantly, the visualization of mitochondrial viscosity has been achieved in both fatty liver and tumor living mice for the first time. This work not only advances the study of the relationship between disease and mitochondrial viscosity but also opens up an efficient way for diagnosing mitochondrial viscosity related diseases.