<p>A MWCNT-supported PtRhFeCoMo high-entropy alloy (HEA) nanocomposite was synthesized via a facile coprecipitation method. The obtained nanocomposite exhibited excellent oxidase-like and peroxidase-like activities, enabling the oxidation of colorless TMB to blue _oxTMB in the presence and absence of H₂O₂. Based on its strong peroxidase-like activity, an acetylcholinesterase (AChE)-mediated turn-on colorimetric sensing platform was constructed for the detection of trichlorfon. In this system, trichlorfon inhibits AChE activity, thereby suppressing the enzymatic generation of thiocholine. Since thiocholine can quench the catalytic activity of the PtRhFeCoMo/MWCNTs nanocomposite, the absence of thiocholine leads to restoration of TMB oxidation, producing a turn-on colorimetric signal. The proposed sensor exhibited a wide linear range (5–1000 ng·mL⁻¹), a low detection limit (1.1 ng·mL⁻¹), excellent selectivity, and satisfactory anti-interference capability. Furthermore, the method was successfully applied to detect trichlorfon in tap water and apple samples with satisfactory recoveries (98.9%–105.1%). This work not only presents a reliable and efficient strategy for trichlorfon monitoring but also demonstrates the great potential of HEA-based nanozymes in developing advanced biosensors.</p> Graphical abstract <p></p>