β-site amyloid precursor protein cleaving enzyme 1 (BACE1) is the rate-limiting enzyme in the formation of β-amyloid peptides (Aβ), which are important in the development of Alzheimer's disease. It has been demonstrated that albiflorin can alleviate Aβ aggregation. However, the mechanism behind this effect is unknown. In our previous work, albiflorin and its four analogues were isolated from Paeonia lactiflora Pall. This study aims to investigate the relationship of these four compounds with BACE1. Our results show that the albiflorin analogue 4-O-galloylalbiflorin (4-O-GA) had the strongest BACE1 inhibitory effect and was also selective for BACE1. Furthermore, docking study of BACE1 with these analogues yielded similar results, whereby 4-O-GA treatment caused the greatest decreases in both the generation of Aβ_1–42 and Aβ_1–40 and in the expression of BACE1 and HIF-1α. Our findings indicate that 4-O-GA exerted significant and selective inhibitory activities against BACE1, potentially providing a useful agent for developing BACE1 inhibitor drugs.