A sensitive, fast and cheap sensor for quantitative determination of carbaryl pesticide using amperometric acetylcholinesterase (AChE) biosensor based on prussian blue (PB)-chitosan (CHIT) hybrid film was reported. This biosensor was fabricated by firstly electrodepositing PB-CHIT hybrid film on glassy carbon electrode (GCE) and subsequently assembling AChE on PB-CHIT hybrid film. The PB-CHIT hybrid film exhibited a good biocompatibility with AChE and effectively immobilized AChE on it. Nanosized PB uniformly distributed in the PB-CHIT hybrid films and showed a good electrocatalytic activity toward oxidation of thiocholine, which was a product from the hydrolysis of acetylthiocholine catalyzed by AChE. The oxidation potential of thiocholine was decreased from 0.68 V to 0.32 V and accordingly the sensitivity of biosensor was largely improved. The inhibition of carbaryl on the activity of AChE was proportional to carbaryl concentration in the ranges from 0.01 to 0.4 mu M and from 1.0 to 5.0 mu M with the correlation coefficients of 0.9996 and 0.9997, respectively. The detection limit was calculated to be about 3 nM. The biosensor provided a new promising tool for pesticide analysis. (C) 2011 Elsevier Ltd. All rights reserved.