One-dimensional bimetallic Pd@Au core-shell nanorods (NRs) with uniform Au shell were synthesized and applied to construct a highly sensitive acetylcholinesterase (AChE) biosensor for the detection of organophosphate pesticides (OPs). The Pd@Au core-shell nanorods with excellent conductivity, pure contact surface and good biocompatibility can provide an extremely suitable area for the adhesion of AChE. Due to these characteristics, the constructed sensor exhibits a superior performance. The biosensor was prepared via a simple procedure and its optimization studies were discussed. Under optimal conditions, the inhibition of paraoxon (as a model compound for OPs) in linearly proportional to its concentration in the range of 3.6pM–100nM, with the detection limit of 3.6pM (calculated for 10% inhibition). The KMapp value of the fabricated enzyme electrode was calculated to be 0.207mM. The application of this biosensor in the analysis of real samples was also evaluated. Moreover, the prepared biosensor possessed higher sensitivity and acceptable reproducibility and stability, showing great potential in the detection of OPs.