In this work, a nanoporous polyaniline–nanocrystalline zeolite organic–inorganic hybrid material was synthesized. An acetylcholinesterase electrochemical biosensor based on a polyaniline–zeolite nanocomposite material was fabricated for the sensitive detection of neurotransmitter acetylcholine and the resultant biosensor was further employed for the detection of toxic organophosphate pesticides. The results demonstrate that the polyaniline–nanocrystalline zeolite based acetylcholinesterase biosensor exhibited much higher activity when compared to conventional polyaniline. The nanoporous polyaniline–nanocrystalline zeolite based biosensor exhibited good stability, sensitivity, linear range, and limit of detection. The analytical performance of the developed biosensor was demonstrated in the determination of these pesticides in different real samples (apple, cabbage, tap water, and river water) with satisfactory recovery.