Abstract: Using diphenylamine as raw material, a viscosity-sensitive fluorescent probe of 2-2-(5-diphenylamine-thienyl) ethenyl-1, 3 3-trimethyl-H-indole iodonium salt was synthesized by the substitution reaction, salt formation reaction and Knoevenagel reaction in turn. Different ratios of water and glycerol were mixed to simulate the different viscosity environments, and the spectral properties of the probe in different viscosity enviroments were studied. The cytotoxi-city and localization of the probe were evaluated by cytotoxicity and colocalization experiments respectively. The changes of mitochondrial viscosity during apoptosis were monitored by this probe. The results show that the fluorescence intensity and fluorescence lifetime of the probe are sensitive to viscosity, and there is a good linear relationship between the fluorescence signals and the environmental viscosity in a certain range. The probe is non-cytotoxic, and is enriched into mitochondria within live cells. The viscosity of mitochondria increases in the process of apoptosis, and the probe can be used to monitor the viscosity of mitochondria in real time.