As a fundamental physical parameter, viscosity influences diffusion in biological processes. The changes in intracellular viscosity leads to the occurrence of relevant diseases. Monitoring changes in cellular viscosity is important for distinguishing abnormal cells in cell biology and oncologic pathology. Herein, we devised and synthesized a viscosity sensitive fluorescent probe LBX-1. LBX-1 showed high sensitivity, providing a large Stokes shift as well as an enhancement of fluorescent intensity (16.1-fold) from methanol solution to glycerol solution. Furthermore, the probe LBX-1 can localize in mitochondria because of the ability of the probe to penetrate the cell membrane and accumulate in mitochondria. These results suggested that the probe could be utilized in monitoring the changes of mitochondrial viscosity in complex biological systems.