Exploring new fluorescent organic dyes with mitochondria-targeted and viscosity-sensitive functions attracts extensive attention because they may provide access to a better understanding of the microenvironments of the subcellular level via biological imaging. Herein a terpyridyl functionalized rhodamine dye (TRMe) has been facilely designed and prepared, which shows a highly selective fluorescence enhancement response to viscosity (1.0–1238.0 cp) in water-glycerol mixtures with the increased glycerol content. In addition, TRMe can respond to viscosity enhancement over a wide pH range (1.0–10.0) and presents low cell toxicity. Besides, a good linearity between the viscosity and fluorescence lifetime of TRMe can be observed. Significantly, TRMe shows excellent mitochondria-targeted performance with a Pearson's colocalization coefficient of 0.958 and can be employed as a tracer for viscosity imaging in Nystatin-stimulated living HeLa cells.