Abstract: In this paper, fourteen new L-proline derivatives were designed and synthesized, and their acetylcholinesterase (AChE) inhibitory activities were also investigated in vitro. New L-proline derivatives were prepared from substituted 2-bromo-1-acetophenones through four-step reaction; and their bioactivities as AChE inhibitors were measured by Ellman spectrophotometry. The results showed that the target compounds had a certain AChE inhibitory activity to in vitro. The bioactivity of compound 8b was the best of them, and its IC₅₀ value was 5.45 μmol·L^-1, which was better than that of rivastigmine. So the acetylcholinesterase inhibitory activities of new L-proline derivatives were worth to be further studied.