•Optimized surface conditions for the immobilization of an expressive amount of active acetylcholinesterase.•The carbaryl pesticide detection follows a Langmuir isotherm with high affinity adsorption constant.•Simple and straightforward methodology to immobilize a high density of active acetylcholinesterase.•Acetylcholinesterase with the active site close to the interface enabling a fast direct electron transfer.•Enzymatic electrochemical biosensor with low LOD and high sensitivity toward the determination of carbaryl pesticide. Efficient and portable sensors are required to monitor and evaluate pesticide contaminants in the environment, food supply and biological fluids. In this work, a study of indigo carmine and dodecyl sulphate doped polypyrrole - gold nanoparticle nanocomposite films revealed changes during the polymerization that optimized the surface conditions for the immobilization of acetylcholinesterase enzyme (from electric eel), leading to improved catalytic performance. The results suggest a similar behavior to AChE from electric ray, as previously reported in a theoretical study, where the active site is orientated closer to the electrode when the enzyme is immobilized on a positively charged surface, promoting faster direct electron transfer. In addition, this interaction allows the immobilization of an expressive amount of active enzymes. The lack of hindrance led to a high binding interaction of the analyte (carbaryl) towards the adsorption sites of the enzyme (Langmuir isotherm constant of 7.39 × 108). This electrochemical enzymatic biosensor presented linear responses ranging from 0.05 to 0.25 ng mL−1 for carbaryl (a wide spectrum insecticide), with a detection limit of 0.033 ng cm2  mL−1, quantification limit of 0.11 ng cm2  mL-1 and sensitivity of -59.5 × 103A cm-2 mL g−1. The biosensor showed good intra-electrode repeatability and inter-electrode reproducibility with the relative standard deviation of 1.8 % and 3.7 % towards thiocholine oxidation and carbaryl pesticide detection, respectively. The biosensor stability improved when stored at lower temperature (-15 °C).