We have developed Lyso-V, the first fluorescent probe of lysosomal viscosity. Because of its lysosome-actived fluorescence characteristics, Lyso-V has proved to be an ideal lysosomal tracer with high spatial and temporal resolution under laser confocal microscopy. More importantly, Lyso-V shows its practical applicability in real-time quantification of lysosomal viscosity changes in live cells through fluorescence lifetime imaging microscopy.