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In this study, a novel, cost-effective, and environmental-friendly acetylcholinesterase (AChE)-based electrochemical biosensor was developed and utilized for paraoxon-ethyl (PE) detection. In this biosensor, cerium oxide (CeO2) and poly(3,4 ethylenedioxythiophene):poly(styrenesulfonate) (PESOT:PSS) modified paper electrode was fabricated by a simple dip-coating method. The synergistic effect of CeO2 and PEDOT:PSS enhance the performance of AChE-based biosensor in terms of sensitivity and selectivity. The electrical conductivity and stability of the fabricated electrode are further improved with the treatment of ethylene glycol (EG). EG decreases the coulombic interactions between PEDOT and PSS molecules, causes polymer chain reorientation, and improves the non-covalent interaction between cellulose molecules and PEDOT, which further leads to higher conductivity. All electrochemical results validate that the biosensor shows good selectivity (Kselectivity < 1), high sensitivity of 52.10 µA/nM, and a low detection limit of 0.12 nM. The accuracy and applicability of the proposed biosensor were also checked with two real samples. All results validate that the fabricated paper electrode can be a promising alternative to other conventional electrodes. [ABSTRACT FROM AUTHOR]