Peroxynitrite anion (ONOO -) is a crucial reactive species in pathological and physiological processes, and the elevated levels of ONOO - is concerned with various diseases mechanism. Researches demonstrated its significant contribution to the development of Alzheimer’s disease (AD). In order to elucidate the role of ONOO - in the evolution of AD, herein an activated fluorescent probe A-Hcy-Br was designed initiated the reaction with probe A-Hcy-Br using ONOO -. The probe has extraordinary specificity, sensitivity, and a rapid responsiveness for ONOO -. In addition, due to the excellent mitochondrial targeting ability of probe A-Hcy-Br, it enables effective monitor of brain cells during ONOO - oxidative stress in nerve cells. It is worth noting that A-Hcy-Br displayed well-behaved near-infrared emission and blood-brain barrier (BBB) permeability, encouraging ONOO - expression imaging in the brain. Moreover, the excellent afterglow imaging ability of A-Hcy-Br was successfully used for afterglow imaging in AD mice brain. A-Hcy-Br is further applicable for real-time fluorescence/afterglow monitoring of the therapeutic effect of the AD treatment drug donepezil hydrochloride, providing visual guidance for AD’s drug therapy.