Rational design of water-soluble probes for mitochondrial viscosity in practical biological applications remains a challenge. Herein, we described a novel hydro soluble benzothiazole salt derivative MitoSN, which exhibits specifically response and singular sensitivity to the mitochondria viscosity in living Hela cells. MitoSN displays an excellent fluorescence enhancement (ca. 35-fold) with the increase of the viscosity in the water-glycerol system. Moreover, confocal microscopy indicates that MitoSN is sensitive to the local viscosity and selectively stains mitochondria, the body of zebrafish as well. Importantly, MitoSN is capable to identify the viscosity difference of mitochondria in normal and nystatin treated Hela cells. The work provides a useful tool to monitor the changes of viscosity in the mitochondrial microenvironment. (C) 2018 Elsevier B.V. All rights reserved.