In this work, the polyaniline/reduced graphene oxide (PANi/rGO) bilayer was directly electrodeposited on carbon screen-printed electrodes (SPE). Some details in growth of PANi/rGO bilayer were revealed from cyclic voltammograms and X-ray photoelectron spectra. The growth of stacked rGO film at high compactness on the electrode surface is mainly accompanied with reduction of epoxy functional groups at basal planes of graphitic flakes. The asgrown rGO layer with abundent hydroxyl functional groups at basal planes is preferable to attract intrinsic fibrillar-like PANi polymer chains in protonated aqueous media. The as-prepared PANi/rGO hybrid bilayer has shown good conductivity, high porosity, good adhesion to biomolecules, and fast electron transfer rate (increased by 3.8 times). Herein, PANi/rGO film has been further utilized to develop disposable acetylcholinesterase sensors able to detect acetylthiocholine (ATCh) with apparent Michaelis-Menten constant of 0.728 mM. These sensors provide a very promising technical solution for in-situ monitoring acetylthiocholine level in patients with neuro-diseases and determination of neuro-toxins such as sarin and pesticides.