Abstract: Objective To screen the peptide inhibitor of acetylcholinesterase (AChE) from 12-mer random phage display peptide library. Methods Human AChE was used as the target to screen its binding peptides from 12-mer random phage display peptide library. The positive phage clones were isolated after three rounds of biopanning followed then by sequence analysis and their activity evaluation. Results Six positive phage clones binding to human AChE were obtained, and 4 of them sharing the conservative sequence W(S/P)HY inhibited the enzyme activity of AChE. Conclusion Acquisition of AChE inhibitor from phage display library provides clues for designing peptide inhibitors of AChE.