In this paper, a novel nanocomposite is chemically synthesized by reinforcing Ag-doped CuO (Ag@CuO) nanoparticles into the matrix of polyaniline (PANI). This nanocomposite is used as a sensing platform for paraoxon-ethyl (PE) detection. The homogeneous distribution of Ag@CuO nanoparticles onto the PANI matrix provides a smooth and dense surface area, further accelerating the transmission of electrons. The synergistic effect of Ag@CuO and PANI matrix is responsible for the outstanding conductivity, biocompatibility, and catalytic power of the proposed biosensor. For the biosensor fabrication, the nanocomposite is electrophoretically deposited onto the surface of the ITO glass substrate and then the acetylcholinesterase (AChE) enzyme is immobilized onto the fabricated electrode surface by using glutaraldehyde as a crosslinking agent. Under all optimized detection conditions, the proposed biosensor (AChE/Ag@CuO/PANI/ITO) shows a broad linear range from 5 to 100 pM with a low detection limit (LOD) of 11.35 pM. All the electrochemical results confirm the high sensitivity (0.5536 μA (pM)−1 cm−2), high selectivity, good reproducibility, and acceptable stability of the proposed biosensor. Moreover, the proposed biosensor is successfully applied for paraoxon-ethyl (PE) detection in three real samples. • Ag@CuO/PANI based nanocomposite is used as sensing platform for paraoxon-ethyl detection. • The incorporation of Ag@CuO into PANI matrix enhances the surface area, catalytic power and conductivity of nanocomposite • The electrophoretic deposition of Ag@CuO/PANI nanocomposite onto ITO glass substrate enhances electrochemical performance • The biosensor shows high linear range (5–100 pM), high sensitivity (0.55 μA (pM)-1 cm-2) and low detection limit (11.35 pM) • Validation with real samples (banana, tomato and soil) shows its good accuracy and applicability for pesticide detection [ABSTRACT FROM AUTHOR]