A solid contact biosensor for Acetylcholine (ACh) based on host-guest interactions and potentiometric transduction has been designed and characterized. The biomimetic man-tailored host was synthesized using methacrylic acid as a functional monomer, ethylene glycol di-methacrylate as a crosslinker in the presence of benzoyl peroxide as an initiator. The imprinted beads were dispersed in 2-nitrophenyloctyl ether and entrapped in a poly(vinyl chloride) matrix. Slopes and detection limits are 55.2-59.6 mV decade-1 and 0.65-1.31 μg mL-1, respectively. Significantly, improved accuracy, precision, good reproducibility, long-term stability, selectivity and sensitivity were offered by these simple and cost-effective potentiometric biosensors. A tubular version was further developed and coupled to a flow injection system for acetylcholine determination. This simple and inexpensive flow injection analysis manifold, with a good potentiometric detector, enabled the analysis of ∼30 samples h-1 without requiring pretreatment procedures. An average recovery of 98.3 % and a mean standard deviation of 1.1% were obtained. The sensors were used to follow up the decrease of a fixed concentration of ACh+ substrate as a function of acetylcholinesterase (AChE) activity under optimized conditions of pH and temperature. A linear relationship between the hydrolysis initial rate of ACh+ substrate and enzyme activity hold 0.01- 5.0 IU L-1 of AChE enzyme.