SERTlight is a fluorescent agent that specifically labels serotonergic neuron cell bodies, dendrites, and axon projections as the fluorescent substrate for the serotonin transporter (SERT). SERTlight is orthogonal in terms of optics, pharmacology, and operations with various genetically encoded sensors, allowing for multiplex imaging. It facilitates the marking of distal 5HT axon projections using GRAB5HT sensors and simultaneously enables imaging of the release of endogenous 5HT, offering a new multifunctional molecular tool for studying the serotonergic system.