Norepinephrine (NE) is a key biogenic monoamine neurotransmitter involved in a wide range of physiological processes. However, its precise dynamics and regulation remain poorly characterized, in part due to limitations of available techniques for measuring NE in vivo. Here, we developed a family of GPCR activation-based NE (GRABNE) sensors with a 230% peak ΔF/F0 response to NE, good photostability, nanomolar-to-micromolar sensitivities, sub-second kinetics, and high specificity. Viral- or transgenic-mediated expression of GRABNE sensors was able to detect electrical-stimulation-evoked NE release in the locus coeruleus (LC) of mouse brain slices, looming-evoked NE release in the midbrain of live zebrafish, as well as optogenetically and behaviorally triggered NE release in the LC and hypothalamus of freely moving mice. Thus, GRABNE sensors are robust tools for rapid and specific monitoring of in vivo NE transmission in both physiological and pathological processes. Display omitted Display omitted •GRABNE sensors are genetically encoded GPCR activation-based norepinephrine sensors•GRABNE distinguishes norepinephrine from dopamine with 1,000-fold specificity•The norepinephrine measurements are sensitive, with high spatiotemporal resolution•Norepinephrine dynamics are observed during stressful behaviors in zebrafish and mice Feng et al. develop and validate a pair of genetically encoded GPCR-activation-based norepinephrine sensors, which, for the first time, enable specific in vivo measurement of norepinephrine dynamics during stressful behaviors with high spatiotemporal resolution in zebrafish and mice.