Epilepsy is characterized by oxidative stress in the brain. As the crucial reductive biothiol, cysteine (Cys) directly regulates the occurrence of oxidative stress in the brain. Observations suggest that the decreased cysteine in plasma could potentially serve as a redox biomarker in temporal lobe epilepsy. However, due to the complexity of the brain and lack of appropriate in situ detecting tools, the concentration change and regulation of Cys in epileptic brains remain unclear. Here, we report a near-infrared imaging probe (named Mito-CP) for tracking endogenous Cys in brains of pentylenetetrazole (PTZ)-induced epileptic seizures with high sensitivity and selectivity. Using this probe, we achieved an in situ visualization of the increased Cys in PC12 cells under dithiothreitol stimulation. In addition, Mito-CP was able to real-time monitor Cys fluctuation in lipopolysaccharide-mediated oxidative stress in zebrafish. Ultimately, we directly visualized the precipitous reduction of Cys in epileptic brains for the first time. Mito-CP also revealed the fluctuation of Cys in epileptic brains during the treatment by an antiepileptic drug, curcumin. This work provides an effective tool for Cys imaging in brains and will help to expand the understanding of the pathogenesis of epilepsy.