Epilepsy is a neurological brain disease, and its recurrent seizures are related to the reductive substance-powered antioxidant defense system (ADS). However, until now, there has been no report on the study of in situ antioxidant fluctuation during epilepsy of varying severity. In this work, hydrogen sulfide (H2S) was selected as the model target, a H2S-responsive near-infrared fluorophore was designed and synthesized, and an amphiphilic molecule was synthesized and modified with angiopep-2 peptide at its hydrophilic terminus. A nanobeacon termed as BFPP was prepared by the formation of micelles with the package of the fluorophore. The nanobeacon was sensitive to H2S, with a low detection limit of 17 nM. The H2S fluctuation in cells can be monitored by fluorescence imaging. In addition, angiopep-2 peptide at the surface of BFPP helps it cross the blood-brain barrier, and near-infrared fluorescence improves in vivo imaging. BFPP revealed that H2S was at a moderate level in the normal brain, but its level was obviously elevated during mild epilepsy because of the activation of the ADS while significantly suppressed during severe epilepsy due to neuronal damage. This approach is generally accessible for other targets by altering the responsive fluorophore, with significance for in situ analysis of brain pathology.