Acetylcholinesterase (AChE) activity may be useful biomarker for detecting of organophosphate pesticides (OP). Thus a sensitive biosensor for quantitative determination of OP based on AChE biomonitoring was developed. Multi-walled carbon nanotube (MWCNT)/polyaniline (PANI) composite film was prepared by electrochemical polymerization. The immobilized AChE catalyzed the hydrolysis of acetylthiocholine chloride to produce thiocholine, which engendered an irreversible oxidation peak. The enzyme activity was monitored by measuring the oxidation current of thiocholine and further detection of OP. The developed sensor provided a new promising tool for pesticide analysis and assay of enzyme activity.