Abstract: This study is designed to obtain recombinant human acetylcholinesterase (rhAChE) and apply it in screening acetylcholinesterase inhibitors. The rhAChE was overexpressed in HEK293 cells transfected by plasmid of pCMV-AChE with the cationic liposome and rhAChE was found to be secreted into cell culture medium. AChE activity was assayed according to modified Ellman method to obtain kinetic parameters. IC₅₀ values for donepezil compounds of rhAChE were calculated to determine their activities of inhibition. The results showed that K_m value was 151.9 祄ol·L^-1, donepezil inhibited rhAChE in a mixed competitive-non-competitive way (K_i = 16.03 nmol·L^-1, K_i' = 18.36 nmol·L^-1) and that most new compounds tested exhibited high activities of inhibition on rhAChE. The study suggests that rhAChE is available to be applied in screening AChE inhibitors in vitro.