A mediator-free amperometric biosensor for screening organophosphorus pesticides (OPs) in flow-injection analysis (FIA) system based on anticholinesterase activity of OPs to immobilized acetylcholinesterase enzyme (AChE) has been developed. The enzyme biosensor is prepared by entrapping AChE in Al(2)O(3) sol-gel matrix screen-printed on an integrated 3-electrode plastic chip. This strategy is found not only increase the stability of the embedded AChE, but also effectively catalyze the oxidative reaction of thiocholine, making the Al(2)O(3)-AChE biosensor detects the substrate at 0.25V (versus Ag/AgCl), hundreds mini-volt lower than other reported mediator-free ones. The Al(2)O(3)-AChE biosensor is thus coupled to FIA system to build up a simple and low-cost FIA-EC system for screening OPs in real samples. A wide linear inhibition response for dichlorvos, typical OP, is observed in the range of 0.1-80muM, corresponding to 7.91-84.94% inhibition for AChE. The detection limit for dichlorvos is achieved at 10nM in the simulated seawater for 15min inhibiting time, which allows the biosensor quantitatively detects the ecotoxicological effect of the real samples from the seaports in eastern China, where the OPs pollution is confirmed by GC-MS.