Acetylcholinesterase inhibitors (AChEIs) are considered to be one of the most successful therapeutic strategies in the treatment of Alzheimer's disease (AD). To enlarge the scale of chemical scaffolds served as AChEIs, a compound collection containing 1280 approved drugs by the U. S. food and drug administration (FDA) was screened. Six drugs, including Alfuzosin, Tandutinib, Dyclonine, Nefazodone, Miconazole and Mesoridazine exhibited potent inhibitory effects on acetylcholinesterase (AChE). The binding mode indicated their “dual site binding” manner, which targeted the catalytic site (CAS) and peripheral anionic site (PAS) simultaneously. Considering that approved drugs have proper physicochemical properties and good safety, these drugs provided us with good starting point to further design selective and potent AChEIs with novel scaffold and good drug-like ability.