Organophosphate and carbamate pesticides are powerful neurotoxins that impede the activity of cholinesterase enzyme leading to severe health effects. This study reports the development, characterization, and application of acetylcholinesterase (AChE) biosensors based on a gold electrode modified with mercaptobenzothiazole (MBT) self‐assembled monolayer and either poly(o‐methoxyaniline) (POMA) or poly(2,5‐dimethoxyaniline) (PDMA) in the presence of polystyrene sulfonic acid (PSSA). The pesticide biosensors were applied in the aqueous phase detection of diazinon and carbofuran pesticides using Osteryoung square wave voltammetry (SWV) and differential pulse voltammetry (DPV) at low frequencies. The results of the study showed that up to 94% inhibition of the MBT‐polyaniline‐based biosensors can be achieved in sample solutions containing 1.19 ppb of these neurotoxin pesticide compounds. Both Au/MBT/PDMA‐PSSA/AChE and Au/MBT/POMA‐PSSA/AChE biosensors exhibited low detection limits, which were calculated using the percentage inhibition methodology. The Au/MBT/POMA‐PSSA/AChE biosensor exhibited lower detection limits of 0.07 ppb for diazinon and 0.06 ppb for carbofuran than did the Au/MBT/PDMA‐PSSA/AChE sensor system that had detection limits values of 0.14 ppb for diazinon and 0.11 ppb for carbofuran. The average sensitivity of the pesticide biosensor systems is 4.2 µA/ppb. A combination of the high sensor sensitivity and low detection limits means that it will be possible to deploy the polyaniline‐based sensor systems as alarm devices for carbamate and organophosphate pesticides.