Mitochondrial viscosity affects metabolite diffusion and mitochondrial metabolism and is associated with many diseases. However, the accuracy of mitochondria-targeting fluorescent probes in measuring viscosity is unsatisfactory because these probes can diffuse from mitochondria during mitophagy with a decreased mitochondrial membrane potential (MMP). To avoid this problem, by incorporating different alkyl side chains into dihydroxanthene fluorophores (denoted as DHX), we developed six near-infrared (NIR) probes for the accurate detection of mitochondrial viscosity, and the sensitivity to viscosity and the mitochondrial targeting and anchoring capability of these probes increased by increasing the alkyl chain length. Among them, DHX-V-C12 had a highly selective response to viscosity variations with minimum interference from polarity, pH, and other biologically relevant species. Furthermore, DHX-V-C12 was used to monitor the mitochondrial viscosity changes of HeLa cells treated by ionophores (nystatin, monensin) or under starvation conditions. We hope that this mitochondrial targeting and anchoring strategy based on increasing the alkyl chain length will be a general strategy for the accurate detection of mitochondrial analytes, enabling the accurate study of mitochondrial functions.