Serotonin (5-HT) is a phylogenetically conserved monoamine neurotransmitter modulating important processes in the brain. To directly visualize the release of 5-HT, we developed a genetically encoded G-protein-coupled receptor (GPCR)-activation-based 5-HT (GRAB) sensor with high sensitivity, high selectivity, subsecond kinetics and subcellular resolution. GRAB detects 5-HT release in multiple physiological and pathological conditions in both flies and mice and provides new insights into the dynamics and mechanisms of 5-HT signaling.