Acetylcholine (ACh) was detected by using electrochemical deposition of Ni on an electrode. The electrochemical characteristics of the Ni-modified electrodes depend on the fabrication conditions such as the reducing current density and the temperature of the electrolyte. The effect of the reducing current density on the electrode performance is more obvious than that of the temperature of the deposition electrolyte. The optimal conditions for the Ni-modified electrodes are for reducing the and a temperature of the deposition electrolyte of 70°C. The surface morphology, roughness, and electrochemical characteristics of the Ni-modified electrode are analyzed by scanning electron microscopy, atomic force microscopy, and the linear sweeping potential method. The /NiOOH oxidation layers on the freshly prepared Ni-modified electrode are generated by repeating the cyclic voltammetry methods for 20 cycles until the oxidation layers approach stability in the alkaline solution. The optimal ACh sensing potential is 0.45 V (vs. Ag/AgCl). The response current of ACh was linear from to 500 ppm with a sensitivity of 0.182 μA/ppm. The reproducibility of the response current for sensing ACh is good. The electrochemical activities of neuron transmitters are .