The establishment of an objective and effective approach is imperative for depression diagnosis and anti-depressant efficacy evaluation, which might eliminate the drawbacks of current clinical diagnoses that rest on subjective scales. Oxidative stress may lead to depression with an excess of oxidants, but correspondingly, reductants play essential roles in retaining redox homeostasis, one of which is mitochondrial cysteine (Cys). Therefore, real-time and in situ monitoring of mitochondrial Cys fluctuations in the mouse brain is expected to achieve effective diagnosis and pharmacodynamic evaluation of depression. Herein, we developed a near-infrared fluorescence imaging probe (CSS) for the detection of mitochondrial Cys in the mouse brain. CSS is responsive to changes in the level of Cys at 705 nm and has high sensitivity, selectivity, temporal–spatial resolution and deep tissue penetration. Utilizing the probe, we revealed that the concentration of mitochondrial Cys decreased in the brains of mice with depression. Importantly, in the brains of depressed mice with three antidepressants, varying levels of Cys were observed, indicating different antidepressant efficacies. Altogether, this work affords a convincing strategy for the precise diagnosis of depression and pharmacodynamic evaluation of antidepressants.