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Acetylcholine (ACH) is one of the excitatory neurotransmitter in the human body. It is the most abundant neurotransmitter responsible for triggering the activation of postsynaptic neurons, leading to an excitatory response. ACH plays a crucial role in various physiological processes, including muscle contraction, autonomic nervous system regulation, and cognitive functions such as learning and memory. In this study, an electrochemical sensor was prepared based on WO3 nanorods modified glassy carbon electrode for the detection of ACH. The WO3 nanorods provided excellent properties for the electrochemical determination of ACH. The proposed sensor exhibited a wide linear detection range of (0.1 to 400.0 µM) and a low detection limit of 0.025 µM for ACH. These results demonstrate the sensor's high sensitivity in detecting this important neurotransmitter. In addition the developed sensor showed good ability for ACH determination in real samples. This study offers an innovative strategy for the electrochemical detection of ACH, showcasing the potential of nanomaterials in the development of advanced sensing technologies.