Elucidating the intrinsic relationship between disease and mitochondrial viscosity is crucial for early diagnosis. However, current mitochondrial viscosity fluorescent probes are highly dependent on mitochondrial membrane potential (MMP) and are sensitive to other mitochondrial microenvironment parameters. To address these issues, a mitochondria-targeting MMP-independent and viscosity exclusive near-infrared (NIR) fluorescent probe, ACR-DMA, was developed. ACR-DMA consists of thiophene acetonitrile as the skeleton and viscosity-sensitive unit, a pyridinium cation for the mitochondria-targeting group, and a benzyl bromide subunit for mitochondrial immobilization. It is very sensitive to viscosity and shows significant "turn-on" fluorescence behavior at 710 nm with a more than 150-fold fluorescence intensity increase. Furthermore, ACR-DMA can be firmly immobilized in mitochondria and can monitor viscosity changes induced by nystain, monensin, and lipopolysaccharide. Additionally, it was successfully used to visualize mitochondrial viscosity changes resulting from tumors, inflammation, and drug-induced acute kidney injury, revealing the relationship between viscosity and disease both in vitro and in vivo. ACR-DMA is expected to be a promising candidate for diagnosing mitochondrial viscosity-related diseases.