A novel fluorescent sensor, iGluSnFR, was developed to visualize glutamate neurotransmission in vivo. This sensor, constructed from E. coli GltI and circularly permuted GFP (cpGFP), exhibits high signal-to-noise ratio and fast kinetics, making it suitable for in vivo imaging. The sensor was optimized through structural analysis and linker optimization, resulting in a maximum fluorescence change of 4.5 (ΔF/F)max. iGluSnFR was validated in various models, including hippocampal cultures, pyramidal neurons, mouse retina, worms, and zebrafish, where it detected glutamate release events and correlated with electrophysiological responses. In mouse forelimb motor cortex, iGluSnFR revealed task-dependent single-spine activity during running. The sensor was also tested in C. elegans, where it detected glutamate input and calcium signals in AVA neurons, confirming its functionality in vivo. iGluSnFR demonstrated high sensitivity and specificity for glutamate detection, with applications in both in vitro and in vivo imaging. It was used to study synaptic activity in the optic tectum of zebrafish and to monitor glutamate transients in the mouse retina. The sensor showed robust performance in various cellular compartments, including dendrites, spines, and somata, and was compatible with two-photon microscopy. iGluSnFR provided a reliable tool for studying glutamate dynamics in intact neural circuits, offering high spatial and temporal resolution for neurotransmitter release analysis. The sensor's ability to detect glutamate transients in awake, behaving animals makes it a valuable tool for neurobiological research.A novel fluorescent sensor, iGluSnFR, was developed to visualize glutamate neurotransmission in vivo. This sensor, constructed from E. coli GltI and circularly permuted GFP (cpGFP), exhibits high signal-to-noise ratio and fast kinetics, making it suitable for in vivo imaging. The sensor was optimized through structural analysis and linker optimization, resulting in a maximum fluorescence change of 4.5 (ΔF/F)max. iGluSnFR was validated in various models, including hippocampal cultures, pyramidal neurons, mouse retina, worms, and zebrafish, where it detected glutamate release events and correlated with electrophysiological responses. In mouse forelimb motor cortex, iGluSnFR revealed task-dependent single-spine activity during running. The sensor was also tested in C. elegans, where it detected glutamate input and calcium signals in AVA neurons, confirming its functionality in vivo. iGluSnFR demonstrated high sensitivity and specificity for glutamate detection, with applications in both in vitro and in vivo imaging. It was used to study synaptic activity in the optic tectum of zebrafish and to monitor glutamate transients in the mouse retina. The sensor showed robust performance in various cellular compartments, including dendrites, spines, and somata, and was compatible with two-photon microscopy. iGluSnFR provided a reliable tool for studying glutamate dynamics in intact neural circuits, offering high spatial and temporal resolution for neurotransmitter release analysis. The sensor's ability to detect glutamate transients in awake, behaving animals makes it a valuable tool for neurobiological research.