Mitochondria, as essential and interesting organelles within the eukaryotic cells, play key roles in a variety of pathologies, and its abnormalities are closely associated with Alzheimer’s disease (AD) and other diseases. Studies have shown that the abnormal of viscosity and concentration of hydrogen peroxide in mitochondria were all associated with AD. Accordingly, the detection of viscosity and hydrogen peroxide in mitochondria has attracted great attention. However, it remains a great challenge to explore a single probe, which can dual-detect the viscosity and H2O2 in mitochondria. Herein, in two ways to prevent the twisted internal charge transfer (TICT) process, we designed and sythesized the first dual-detection fluorescent probe Mito-VH that can visualize viscosity and H2O2 in mitochondria with different fluorescence signals in living cells.