Mitochondrial cysteine (Cys) and viscosity are closely associated with numerous cellular processes and diseases. Developing a method capable of real-time tracking of viscosity and Cys in mitochondria is crucial for understanding cell status and mitochondria-related diseases. In this study, we developed a mitochondria-targeted fluorescent probe (Mito-QP) with a large Stokes shift of 171 nm for detecting Cys and viscosity. The acryloyl group enabled Mito-QP specific response to Cys with high sensitivity. The probe exhibited significant red fluorescence enhancement with increasing viscosity due to restricted intramolecular rotation. Additionally, Mito-QP showed low cytotoxicity and excellent mitochondrial-targeting ability. The probe was successfully employed to monitor fluctuations in mitochondrial viscosity and Cys in cancer cells and zebrafish. This work provides a convenient and effective sensor for studying mitochondrial dysfunction.