This research presents the construction and optimization of an acetylcholine, ACh, ion-selective electrode, ACh-ISE, aiming to use it for the quantification of the propoxur pesticide. This analytical quantification is based on the evaluation of the acetylcholinesterase enzyme inhibition percentage, %EI, provoked by the pesticide, as a function of propoxur concentration; where the %EI is followed by means of the potentiometric determination of acetylcholine that does not react with the enzyme, using the constructed ACh-ISE. It was found that the ACh-ISE depicted a sub-Nernstian slope of (42.7 ± 2.8) mV decade −1 , a ACh detection limit of 1.2 × 10 −5 mol l −1 , and the selectivity constants, K pot , of 0.03 and 0.24 for Na + and Choline + cations, respectively. K + and Ca 2+ did not interfere. This new method designed to determine propoxur attained an outstanding detection limit of (2.89 ± 0.97) × 10 −17 mol l −1 and a linear interval of (5.5 × 10 −15 -1.0 × 10 −4 ) mol l −1 .