An acetylcholinesterase (AChE)‐lecithin biomimetic structure was constructed at the oil/water interface for the direct determination of fenthion in cyclohexane. Indophenol acetate in oil phase was hydrolyzed by AChE at the two‐phase interface to produce indophenol. Square wave voltammetry was used to monitor the current decrease of substrate to assess AChE activity. The AChE incorporated in the lecithin‐based biomimetic layer possessed higher enzymatic activity and was more sensitive to fenthion inhibition than freestanding AChE. A linear relationship between the inhibition percentage and logarithm of fenthion concentration was obtained in a concentration range from 1 ng/mL to 1 mg/mL.