Ischemic stroke is caused when blood flow to the brain is stopped and is a major cause of death and long term disability across the globe. Excessive release of neurotransmitters is triggered in the brain by ischemia that mediates neuronal damage and causes ischemic injury. In this study, a simple, sensitive, and on-line preconcentration capillary electrophoresis method based on electrokinetic supercharging (EKS) was developed for the determination of the biogenic amines including dopamine (DA), epinephrine (E), and norepinephrine (NE) in C57BL/6 mice brain. Under the optimized conditions, the analytes were concentrated and detected within 10 min. The detection limits for the analytes ranged from 0.42 to 0.57 ng mL(-1) for a mice brain matrix. With the proposed method, the analyses of three neurochemical amines in C57BL/6 mice brain tissue during cerebral ischemic/reperfusion had been performed successfully.