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Organophosphate and carbamate pesticides are powerful neurotoxins that impede the activity of cholinesterase enzyme leading to severe health effects. This study firstly reports the development, characterisation, and application of thick-film acetylcholinesterase (AChE) biosensors based on a gold electrode modified with a mercaptobenzothiazole (MBT) self-assembled monolayer and either poly(o-methoxyaniline) (POMA) or poly(2,5-dimethoxyaniline) (PDMA) in the presence of polystyrene(4-sulphonic acid) (PSSA). The Au/MBT/POMA-PSSA/AChE and Au/MBT/PDMA-PSSA/AChE biosensors were then applied to successfully detect standard organophosphorous and carbamate pesticides in a 0.1 M phosphate buffer, 0.1 M KCl (pH 7.2) solution. Secondly, it reports the construction of the Au/MBT/PANI/AChE/PVAc thick-film biosensor for the determination of certain organophosphate and carbamate pesticide solutions in selected aqueous organic solvent solutions.