Abstract: Endophytic fungi of Polygala sibirica collected from Shuangjiang County of Yunnan Province were isolated and purified with traditional culture method, then these fungi were identified based on ITS sequences. The fermentation extracts of the fungi were screened for acetylcholinesterase inhibitory activity to explore the diversity of endophytic fungi in P. sibirica and their potential for treating Alzheimer 's disease. The findings revealed that a total of 23 strains of endophytic fungi, including 18 from aerial tissues and 5 from the root of P. sibirica, were isolated. These fungi belonged to 13 genera, 11 families, 8 orders, 4 classes and 1 phylum of Ascomycota. The dominant genera were Colletotrichum, Epicoccum and Fusarium. The inhibitory activities of methanol extracts of the 23 strains against acetylcholinesterase(AChE) were determined by modified Ellman's method. At a concentration of 0.5 mg/mL, 12 of the strains showed moderate or high inhibitory effect against AChE(inhibition rate>30%). The strain Fusarium oxysporum PSA008 exhibited the most potent inhibitory effect on AChE with inhibition rate of 75.35%, and other 2 strains(Boeremia exigua PSA002 and Penicillium brefeldianum PSB007) also showed high AChE inhibitory activity(inhibition rate>50%). UPLC-Q-TOF/MS was used to analyze the methanol extracts from rice culture of 3 strains of fungi, including PSA002, PSA008 and PSB007. The results showed that the response intensity and quantity of mass spectrum data points of the 3 samples were apparent and different, and compared with rice medium, there were also differences. The results indicated that the metabolic transformation of these fungi formed certain chemical diversity components. This study can provide basic research materials and a theoretical basis for further exploration of related compounds with AChE inhibitory activity.