The neurotransmitter acetylcholine (ACh) regulates a diverse array of physiological processes throughout the body. Despite its importance, cholinergic transmission in the majority of tissues and organs remains poorly understood owing primarily to the limitations of available ACh-monitoring techniques. We developed a family of ACh sensors (GACh) based on G-protein-coupled receptors that has the sensitivity, specificity, signal-to-noise ratio, kinetics and photostability suitable for monitoring ACh signals in vitro and in vivo. GACh sensors were validated with transfection, viral and/or transgenic expression in a dozen types of neuronal and non-neuronal cells prepared from multiple animal species. In all preparations, GACh sensors selectively responded to exogenous and/or endogenous ACh with robust fluorescence signals that were captured by epifluorescence, confocal, and/or two-photon microscopy. Moreover, analysis of endogenous ACh release revealed firing-pattern-dependent release and restricted volume transmission, resolving two long-standing questions about central cholinergic transmission. Thus, GACh sensors provide a user-friendly, broadly applicable tool for monitoring cholinergic transmission underlying diverse biological processes.