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[Display omitted] • A novel resorufin-based fluorescent probe for selective detection of NE was developed. • Probe displayed rapid response time (<10 min) and high selectivity towards NE. • Probe could detect endogenous NE and exocytosis process by fluorescent imaging in living cells and depression-like behavior mice. Norepinephrine (NE), as an important neurotransmitter, is closely associated with the pathogenesis of anxiety and depressive disorders. Effective monitoring of NE fluctuation aids in the diagnosis of depression and the therapeutic assessment of the antidepressant intervention. The construction of novel fluorescent probes with high specificity towards NE for imaging in depression models is still in demand urgently. In this work, a novel resorufin-based red-emitting fluorescent probe for real-time tracking NE was developed. NE can significantly increase the fluorescence of probe LNE by triggering deprotection of carbonothioate ligand via nucleophilic substitution. The probe LNE demonstrated significant NE selectivity and sensitivity over other analytes in vitro. In addition, probe LNE showed a fast response time (<10 min), and the change in fluorescence signal was positively linked with NE concentration, which could be utilized to track the dysregulation of NE in vivo. More importantly, this powerful probe was successfully employed for real-time visual and imaging of NE in living cells and depression-like behavior animals. [ABSTRACT FROM AUTHOR]