A mono-enzymatic acetylcholinesterase (AChE) amperometric biosensor for organophosphate detection was developed immobilizing the AChE enzyme via glutaraldehyde on a preformed cysteamine self-assembled monolayer (SAM) on gold-screen printed electrodes (Au-SPEs). The enzymatic activity was monitored measuring the enzymatic product, thiocholine, at an applied potential of +400mV vs. Ag/AgCl using ferricyanide in solution as electrochemical mediator. The electrocatalytic activity of ferricyanide towards thiocholine was investigated by using Nicholson–Shain method and finding a second order homogenous rate constant k_s equal to (5.26±0.65)×10⁴M⁻¹s⁻¹. In order to develop a sensitive biosensor, the effect of cysteamine concentration, duration of SAM deposition and AChE concentration were optimized. Using paraoxon as model compound, the biosensor showed a linear range up to 40ppb with a detection limit of 2ppb (10% of inhibition). The biosensor was successfully challenged with drinking water sample demonstrating to be a useful analytical tool for organophosphorus insecticide detection.