© 2018 R. Montes et al. This paper presents the characterization and optimization of biosensors based on graphite-epoxy which incorporates the enzyme acetylcholinesterase (AChE). By means of advanced electrochemical techniques, such as electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV), the characterization and optimization of graphite-epoxy-AChE biosensors have been performed. In order to obtain sensitive electrodes, the optimal composition of the transducer material (graphite-epoxy-enzyme ratio) was studied. The optimization of the conductive particles distribution inside the biomaterial has allowed an improvement of the electrochemical properties. Optimal composition guarantees improving electrochemical properties required, such as high electron-transfer rate, high signal-to-noise ratio, and suitable sensitivity. The optimal biocomposite composition range was obtained between 16% and 17% of graphite and 0.12% of AChE. The biosensors were applied to the analysis of different pesticides, organophosphorus and carbamates, using indirect measurements based on enzymatic inhibition process. These optimized biosensors present detection limit one order of magnitude lower compared to the standard composition (nonoptimized) and allow achieving concentrations lower than the established ones by the pesticides regulation. Finally, spiked tap water samples with pesticides were analyzed with the optimized biosensors.