As a neurotransmitter, norepinephrine (NE) is critical for psychiatric conditions, neurodegenerative diseases, and pheochromocytoma. A real-time and noninvasive method for the detection of NE as a tracer to investigate the NE-relevant disease treatment process is urgently desirable. Herein, we successfully developed a turn-on NE bioluminescent probe (NBP), which was grounded on p-toluenethiol deprotectrf by nucleophilic substitution. Compared with other analytes, the NBP exhibited high sensitivity and selectivity in vitro. More importantly, the NBP provides a promising strategy for in vivo imaging of NE in living animals with noninvasive visualization and real-time features.