We report a screen-printed electrode (SPE) modified with a dispersion of carbon black (CB) and chitosan by drop casting. A cyclic voltammetry technique towards ferricyanide, caffeic acid, hydroquinone, and thiocholine was performed and an improvement of the electrochemical response with respect to bare SPE as well as SPE modified only with chitosan was observed. The possibility to detect thiocholine at a low applied potential with high sensitivity was exploited and an acetylcholinesterase (AChE) biosensor was developed. A dispersion of CB, chitosan, and AChE was used to fabricate this biosensor in one step by drop casting. The enzymatic activity of the immobilized AChE was determined measuring the enzymatic product thiocholine at +300 mV. Owing to the capability of organophosphorus pesticides to inhibit AChE, this biosensor was used to detect these pollutants, and paraoxon was taken as model compound. The enzyme inhibition was linearly related to the concentration of paraoxon up to 0.5 μg L–1, and a low detection limit equal to 0.05 μg L–1 (calculated as 10% of inhibition) was achieved. This biosensor was challenged for paraoxon detection in drinking waters with satisfactory recovery values. The use of AChE embedded in a dispersion of CB and chitosan allowed an easy and fast production of a sensitive biosensor suitable for paraoxon detection in drinking waters at legal limit levels.