〔Abstract〕 Objective To establish a screening system for cholinesterase inhibitors. Methods Acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) were obtained from the brain and serum of SD rats. Ellman colorimetry assay was used to establish an in vitro experimental system for the screening of AChE and BuChE inhibitors. Results The 10–fold dilution of male rat brain homogenate (protein concentration, 1.69 ± 0.10 mg/ mL) and 5–fold dilution of serum were selected as AChE / BUCHE enzyme sources, and the AChE/BuChE inhibitor, tacrine, was used as the reference. It was found that tacrine inhibited AChE and BuChE with an IC50 of 262 nM and 168 nM, respectively, an observation consistent with the literature reports, suggesting that the screening system for cholinesterase inhibitors in vitro has been successfully established. In addition, the activity of AChE enzyme activity in the brain homogenate of female rats was 1.84 times than that of male rats under the same amount of protein. Conclusions AChE and BuChE crude enzymes were extracted from rat brain and serum respectively to construct a sensitive and stable AChE/ BuChE inhibitor screening platform.