ABSTRACT: Acetylcholine (ACh) is a critical neurotransmitter influencing various neurophysiological functions. Despite its significance, there is a lack of quantitative methods with adequate spatiotemporal resolution for recording single exocytotic efflux of ACh. In this study, we present an ultrafast amperometric ACh biosensor enabling electrochemical recording that captures spontaneous bursts of single presynaptic exocytosis events at axon terminals of cholinergic cells with sub-millisecond temporal resolution. Characterization of the recorded amperometric time trace revealed seven distinct current spike types, each displaying variations in both spike shape, time scale and quantities of ACh released through the synaptic vesicle fusion pore. This observation suggests the presence of multiple exocytosis modes at these cells. Quantifying the absolute number of ACh molecules released at single exocytosis events was achieved through sensor calibration using electroanalytical measurements of synthetic lipid vesicles containing varying concentrations of ACh. Notably, the largest quantal release was estimated at approximately 8000 ACh molecules, likely representing full exocytosis, while the fractional release of roughly 5000 ACh molecules correspond to a partial exocytosis mode. Following a local administration of bafilomycin A1, a V-ATPase inhibitor, the cholinergic cells exhibited both a higher frequency and larger quantity of ACh released during exocytosis events. Hence, this ACh sensor introduces means to monitor minute amounts of ACh and investigate regulatory mechanisms at single-cell level, which is vital for understanding healthy brain function, pathologies, and optimizing drug treatment for disorders.