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A novel method for the sensing of acetylcholinesterase (AChE) activity and inhibitor screening based on the formation of metal coordination polymer has been developed. Acetylthiocholine (ATCh) was selected as the substrate. In the presence of AChE, ATCh was hydrolyzed to thiocholine and acetate. Thiocholine interacted with Ag(I) to form a metal coordination polymer. A positively charged perylene probe (probe 1) was employed. The fluorescence of probe 1 was very efficiently quenched by a polyanion [PVS, poly(vinyl sulfonate)]. In the presence of acetylcholinesterase, the positively charged metal coordination polymer newly formed in situ would interact with PVS, probe 1 monomer molecules were released, and a turn on fluorescence signal was detected. The assay is highly sensitive, a limit of detection of 0.04 mU/mL AChE was obtained. The assay is also highly selective, a number of potential interference proteins (enzymes) were tested, and none of them show noticeable interference. Sensing of AChE inhibitor was also demonstrated. Our assay is fairly simple and inexpensive. We envision that it could be used for the sensitive detection of other hydrolytic enzyme activities with properly selected substrates and for the screening of potential inhibitor drugs.