Acetylcholine (ACh) present inside the brain has ability to alter neuronal activation, influencing transmission of synapse by inducing neurons causing the enhancement in the ability of neuronal networks inside the brain. In result, ACh modifies the response of internal and external environment and perform function as a neuromodulator. This empowers the plan of a sensor particular for ACh that is effectively brought into biological frameworks. The work described here, an acetylcholinesterase (AChe) based sensor which is the most promising tool for analysis of neuromodulator level in biological samples. The prepared biosensor interface is based on co-immobilization of the AChe and choline oxidase (Cho) onto palladium nanoparticles (Pd-nano) adsorbed over molybdenum disulfide (MoS2) nanostructures electrodeposited on the surface of gold electrode (Au-ET). The sensor had excellent linear range of 1.0 nM to 10 niM with 1.0 nM as limit of detection. The novel AChe-Cho/Pdnano/MoS2@Au-ET shows a tremendous potential for applications in medical research and clinical diagnosis. The novel biosensor formed has good accuracy, precision and reliability hence, it might provide a new outlook for the detection and recognition of ACh in real serum samples.