The authors describe the development and characterization of an intensity-based glutamate-sensing fluorescent reporter (iGluSnFR) for visualizing glutamate neurotransmission in vivo. iGluSnFR was engineered to maximize fluorescence change and validated for its utility in visualizing glutamate release by neurons and astrocytes in various neurological systems. The sensor exhibited high signal-to-noise ratio and fast kinetics, and correlated with electrophysiological responses. iGluSnFR was successfully used in hippocampal cultures, mouse retina, worms, zebrafish, and mouse motor cortex to detect single field stimulus-evoked glutamate release events, tonic glutamate signaling, and task-dependent single-spine activity during running. The sensor's performance in different species and experimental conditions demonstrates its broad applicability for studying glutamate neurotransmission.The authors describe the development and characterization of an intensity-based glutamate-sensing fluorescent reporter (iGluSnFR) for visualizing glutamate neurotransmission in vivo. iGluSnFR was engineered to maximize fluorescence change and validated for its utility in visualizing glutamate release by neurons and astrocytes in various neurological systems. The sensor exhibited high signal-to-noise ratio and fast kinetics, and correlated with electrophysiological responses. iGluSnFR was successfully used in hippocampal cultures, mouse retina, worms, zebrafish, and mouse motor cortex to detect single field stimulus-evoked glutamate release events, tonic glutamate signaling, and task-dependent single-spine activity during running. The sensor's performance in different species and experimental conditions demonstrates its broad applicability for studying glutamate neurotransmission.