Acetylcholine is a neurotransmitter, which is located at the intersections of the nerve and muscles in the lymph nodes of the internal organs motor systems and in various parts of the central nervous system. A decrease of acetylcholine in brain is associated with Alzheimer‘s disease. That is why it is an important agent for this disease. In this study, a bienzymatic biosensor system with acetylcholine esterase and choline oxidase was prepared with carbon paste electrode modified with carbon nano Dot-(3-Aminopropyl) triethoxysilane (CDs-APTES) for determination of the amount of acetylcholine. Acetylcholine esterase and choline oxidase enzymes were immobilized onto a modified carbon paste electrode by cross-linking with glutaraldehyde. Determination of acetylcholine was carried out by the oxidation of enzymatically produced H2O2 at 0.4 V versus Ag/AgCl. The effect of temperature, pH, and substrate concentration on the acetylcholine response of the prepared biosensor was investigated. In addition, the optimum CDs-APTES amount, the linear operating range of the biosensor, and the interference effect were also investigated.