Acetylcholine (ACh), the major neurotransmitter secreted by cholinergic neurons, is widely found in the peripheral and central nervous systems, and its main function is to complete the transmission of neural signals. When cholinergic neurons are impaired, the synthesis and decomposition of ACh are abnormal and the neural signalling transition is blocked. To some extent, the concentration changes of ACh reflects the occurrence and development of many kinds of nervous system diseases, such as Alzheimer's disease, Parkinson's disease, Myasthenia gravis and so on. Thus, researches of the physiological and pathological roles and the tracking of the concentration changes of ACh in vivo are significant to the prevention and treatment of these diseases. In the paper, the pathophysiological functions and the comprehensive research progress on detection methods of ACh are summarized. Specifically, the latest research and related applications of the optical and electrochemical biosensors are described, and the future development directions and challenges are prospected, which provides a reference for the detection and applications of ACh.