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.