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Nowadays, pesticide residues constitute an increasing public health concern. Cholinesterases, acetylcholinesterase, and butyrylcholinesterase, are reported to be involved in detoxification processes owing to their capability of scavenging organophosphates and carbamates. Thus, these enzymes are targeted for the discovery of sensors aiming at detecting pesticide residues. In recent years, cholinesterase-based biosensors have attracted more and more attention in the detection of pesticides. Herein, this review describes the recent progress on the engineering of cholinesterases and the development of the corresponding sensors that could be used for the detection of organophosphorus pesticide residues.