Acetylcholinesterase (AChE) inhibition is a significant strategy for preventing Alzheimer's disease (AD) and neurodegenerative diseases. In this study, a dereplication system was utilized to rapidly identify and characterize acetylcholinesterase-interacting compounds by comparing UPLC-MS/MS profile screening approach and molecular docking analysis, derived from the extracts and fractions of the stem of Glycosmis parviflora (Sims) Little. Eleven potential AChE inhibitors were isolated and identified from the ethyl acetate extract of G. parviflora, including an undescribed alkaloid (9), namely glybomine D, eight known alkaloids (18), a flavonoid (10), and a phytosterol (11). The inhibitory potential of these compounds against AChE was assessed, with O-methylglycosolone (6), 1,3-dimethoxy-2-hydroxy-10-methyl-9(10H)-acridinone (1), skimmianine (4) and arborine (2), regarded as effective inhibitors, yielding IC50 values of 39.81 mu M, 41.53 mu M, 49.40 mu M, and 59.92 mu M, respectively. Notably, O-methylglycosolone exhibited the highest potency. Four of these potent AChE inhibitors exhibited mixed-type inhibition. However, O-methylglycosolone (6), 1,3-dimethoxy-2-hydroxy-10-methyl-9 (10H)-acridinone (1), and arborine (2) were first reported modulating with acetylcholinesterase activity. Furthermore, molecular docking revealed O-methylglycosolone (6) superior binding affinity (-23.749 kcal/mol) compared to other compounds, mainly by interacting with the peripheral anionic site of AChE, which forms hydrogen bonds and hydrophobic forces may play an important role, interaction with amino acid residues such as Tyr341, Tyr72, Ser293, and Arg296 in the active cavity, which is crucial for effective and selective inhibition of AChE activity. ADMET predictions suggest that arborine (2), skimmianine (4), and O-methylglycosolone (6) demonstrate favorable permeability across the blood-brain barrier, while 1,3-dimethoxy-2-hydroxy-10-methyl-9 (10H)-acridinone (1) exhibits comparatively reduced permeability. These findings highlight the potential of these compounds as natural AChE inhibitors for treating neurodegenerative diseases.