Tremendous efforts have been put into exploring high-entropy alloys (HEAs) for electrocatalysis, which is considered to be a pivotal cornerstone for electrochemical energy conversion. Nevertheless, HEAs have not been employed for electrochemical biosensing and the precise construction of low-dimensional architectures for HEAs is still challenging. Herein, ultrathin quinary PtPdRhCuM (M= Mn, Co or Ni) HEA nanotubes (NTs) with noticeable lattice distortion as well as rich defect sites were controllably synthesized by a template-triggered strategy combining galvanic exchange process, co-reduction pathway and the dealloying technology. Owing to the diversity of composition, high mixing entropy, intrinsic electronic conductivity, and complexity of surface structure, the PtPdRhCuM (M= Mn, Co or Ni) HEA exhibits remarkable physicochemical property and serves as an ultrasensitive sensing layer for the determination of aldicarb sulfone. The acetylcholinesterase (AChE)-Nafion/PtPdRhCuMn HEA NTs-based biosensor possessed a wide detection range of 0.45 pM – 45 nM with a low detection limit of 86.5 fM for aldicarb sulfone. Moreover, the established biosensor was applicable to pesticides in real food samples (apple and cucumber) with excellent recoveries (99.87 − 101.49% and 97.95 − 104.56%). This work forms the foundation for employing HEAs as electrode material for widespread applications such as electrochemical aldicarb sulfone sensing.