Acetylcholine (ACh), a neurotransmitter is extensively found in the peripheral and central nervous systems. It is secreted by cholinergic neurons. If these neurons are damaged, the synthesis and decomposition of ACh gets abnormal resulting into blockage of neural signaling transition. The variable level of ACh leads to Alzheimer’s disease, Parkinson’s disease, Myasthenia gravis, and schizophrenia. Hence it is very important to quantify ACh level to diagnose these diseases. Biosensors have great promise as a tool for measuring analytes accurately and quickly. Present review describes the development of various enzymatic and non-enzymatic electrochemical biosensors for detection of ACh. The electrochemical acetylcholine biosensors had shown optimum activity in the pH range 7.0 to 9.0, temperature 30 to 65 °C, response time 3 to 200 s, linearity ranges from10⁻⁵ to 1500 μM and LOD between 0.0001 and 0.3 μM. The sensors had advantages in terms of simplicity, monitoring and requiring no reagents. In future, the use of electrochemical paper analytical devices, lab on a chip (electronic chips) and other nanomaterials based research could be focused on the acetylcholine biosensors.