Acetylcholine (ACh) plays a pivotal role as a neurotransmitter, influencing nerve cell communication and overall nervous system health. Imbalances in ACh levels are linked to neurodegenerative diseases, such as Alzheimer's and Parkinson's. This study focused on developing electrochemical sensors for ACh detection, utilizing graphene oxide (GO) and a composite of reduced graphene oxide and zinc oxide (rGO/ZnO). The synthesis involved modified Hummers' and hydrothermal methods, unveiling the formation of rGO through deoxygenation and the integration of nano-sized ZnO particles onto rGO, as demonstrated by XPS and TEM. EIS analysis also revealed the enhancement of electron transfer efficiency in rGO/ZnO. Cyclic voltammograms of the electrode, comprising the rGO/ZnO composite in ACh solutions, demonstrated prominent oxidation and reduction reactions. Notably, the composite exhibited promise for ACh detection due to its sensitivity, low detection threshold, reusability, and selectivity against interfering compounds, specifically glutamate and gamma-aminobutyric acid. The unique properties of rGO, such as high specific surface area and electron mobility, coupled with ZnO's stability and catalytic efficiency, contributed to the composite's potential in electrochemical sensor applications. This research, emphasizing the synthesis, fabrication, and characterization of the rGO/ZnO composite, established itself as a reliable platform for detecting the acetylcholine neurotransmitter.