Trichlorfon (TCF) is an important chemical for pest and parasite control, but it poses risks to public and environmental health. Thus, the detection of TCF is of great value. In this study, we report a strategy to prepare an AChE-nanoprobe for detecting TCF. Benefiting from the unique and excellent structure of CHO-β-CD, this AChE-nanoprobe can wrap nano-Fe3O4 and covalently bind AChE. As a proof of concept, we prepared an AChE-nanoprobe with a hydrated particle size of 717 nm and a saturation magnetic strength of 64.11 emu/g Fe. The properties were studied in detail. Its catalytic activity was 90 % of that of free AChE, and it had broadened pH, thermal, organic solvents and storage in addition to good reusability, with a residual catalytic activity of 42 % after the 10th cycle and a cumulative catalytic activity 6.9 times higher than that of the free enzyme. The AChE-nanoprobe exhibited good performance in TCF detection; the linear range of TCF was 40–4000 ng/L, and the limit of detection for TCF was 2.8 ng/L. Furthermore, our results suggest that the AChE-nanoprobe has several attractive features, such as easy preparation, outstanding stability and reusability, providing a novel and promising tool for the analysis of TCF.