As an important parameter of the cellular microenvironment, the changes in mitochondrial viscosity are closely related to various life activities. Therefore, the development of fluorescent probes for test the changes of mitochondrial viscosity has great significance. In this study, we developed two fluorescent probes for the detection of the mitochondrial viscosity changes. The probes exhibited different fluorescence intensities at different viscosity based on the twisted intramolecular charge transfer process. The characteristics of high anti-interference performance, wide pH applicability, low cytotoxicity and excellent mitochondrial targeting performance made the probes successfully used to distinguish normal cells from cancer cells, achieving visualization of viscosity changes. Furthermore, probes P1 and P2 can also be used as early diagnosis of tumors in mice and reveal the pathology of tumor development. The probes could be serve as a promising viscosity detection tool for discriminating normal cells and cancer cells in biology-related research.