In this study, we developed a two-photon fluorescent probe for detection of peroxynitrite (ONOO-) near the N-methyl-d-aspartate (NMDA) receptor. This naphthalimide-based probe contains a boronic acid reactive group and an ifenprodil-like tail, which serves as an NMDA receptor targeting unit. The probe displays high sensitivity and selectivity, along with a fast response time in aqueous solution. More importantly, the probe can be employed along with two-photon fluorescence microscopy to detect endogenous ONOO- near NMDA receptors in neuronal cells as well as in hippocampal tissues. The results suggest that the probe has the potential of serving as a useful imaging tool for studying ONOO- related diseases in the nervous system.