The human brain consists of billions of neurons interconnected by chemical synapses, where communication is mediated by the release of neurotransmitters and neuromodulators [1]. Classic neurotransmitters, such as glutamate and γ-aminobutyric acid (GABA), typically mediate rapid point-to-point synaptic transmission by activating ionotropic receptors, sometimes also acting through metabotropic G protein-coupled receptors (GPCRs) [2]. On the other hand, neuromodulators, such as monoamines, neuropeptides, and neurolipids, exert their effects mainly through GPCRs to trigger downstream signaling cascades in a relatively slower and long-range way through diffusion [3], [4]. Neuromodulators play crucial roles in regulating neurotransmission in specific neural circuits, thereby influencing diverse physiological processes, such as cognition, motor control, mood, homeostasis, and learning and memory. Consequently, disruptions in neuromodulatory signaling have been associated with many psychiatric and neurological disorders, including depression [5], [6], schizophrenia [7], [8], Parkinson’s disease [9], [10], and Alzheimer’s disease [11], [12].

Given their fundamental roles in brain function, the ability to monitor neuromodulator dynamics with high precision is crucial for understanding their regulatory mechanisms under both physiological and pathological conditions. However, traditional detection methods, including microdialysis [13], [14], [15], electrochemical [16], [17], [18], and electrophysiological methods [19], are limited by their spatiotemporal resolution and invasiveness, making it difficult to faithfully report the rapid changes of neuromodulators, especially in behaving animals. Over the past few years, the development of genetically encoded fluorescent sensors for neuromodulators has provided a transformative solution to these limitations. These sensors offer high spatial and temporal resolution, molecular specificity, and minimal invasiveness, enabling real-time monitoring of neuromodulator dynamics in vivo across various model organism, including fruit flies, zebrafish, and mice. This review summarizes the design principles, recent progress, and applications of these sensors and discusses future directions of sensor development, aimed at enhancing their utility for neuroscience research.