Epilepsy is a nervous system disease, and seizures are closely related to oxidative stress. Thiols, as the main antioxidant in an organism, play a key role in regulating the redox balance and defending from oxidative stress. As a result of the complexity of the brain structure, there is still a lack of suitable in situ detection methods of thiols to reveal the relationship between epilepsy and thiol level fluctuations. Therefore, by combining picolinate as the new recognition site for thiols, parallel synthesis, and the fluorescence rapid screening method, DCI-Br-3 was developed as a rapid, highly sensitive, and selective probe to monitor thiols in vitro and in vivo. It is worth noting that DCI-Br-3 effectively crossed the blood-brain barrier (BBB) to reveal the negative relationship between the level of thiols and the occurrence of epilepsy and may further provide important information for the prevention and treatment of thiol-related neurological diseases.