Despite the apparent copious fluorescent probes targeting mitochondria, the development of low cytotoxic probes is still needed for improving validation of mitochondrial function assessment. Herein, we report a novel cyanine-based NIR fluorescent probe, T2, which selectively targets mitochondria with significantly low toxicity by modulating the intracellular redox status. Additionally, T2 inhibits oxidative stress-induced cell death in cortical neurons. This study provides new insight into developing low-toxic mitochondrial imaging agents by regulating redox homeostasis.