Objective To explore the protective mechanism of donepezil on Alzheimer"s disease based on Tau protein phosphorylation in serum. Methods From January 2019 to July 2022, samples of two groups of participants were recruited from the Department of Encephalopathy of our hospital. In the first group, 58 patients with mild to severe AD and 20 healthy elderly controls were included, while in the other group, 37 patients with mild to moderate suspected AD were included, of which 18 patients received donepezil treatment for 24 weeks and 19 patients received placebo treatment for 24 weeks. Extracellular vesicles (EVs) derived from neurons were extracted from patients" blood samples, and the levels of Aβ42, P-T181-tau, P-S396-tau, t-tau and NRGN were analyzed by ELISA kit. Results Compared with the control group, the levels of Aβ42, t-tau, P-T181-tau and P-S396-tau in EVs of AD patients increased significantly (P<0.05), and the level of NRGN decreased significantly (P<0.05). In AD patients, the EVs levels of t-tau, NRGN and REST were compared with MMSE (T-Tau: r =-0.267, P < 0.01; NRGN:r =–0.262, P < 0.01; Rest: r =-0.194, P < 0.05) and ADCS-ADL score (t-tau: r =-0.226, P < 0.05; NRGN: r =–0.257, P < 0.01; Rest: r =-0.269, P < 0.01) was negatively correlated with ADAS-cog+ score (t-tau: r = 0.237, P < 0.05; NRGN: r = 0.293, P < 0.01; REST: r = 0.225, P < 0.05). Compared with the placebo group, the levels of Aβ42, t-tau, P-T181-tau and P-S396-tau in donepezil group induced EVs decreased significantly from baseline to the 24th week (P < 0.05). Conclusions The levels of Aβ42, t-tau, P-T181-tau and P-S393-tau in plasma EVs of patients with mild to moderate AD are increased, and the increase of t-tau, NRGN and REST in EVs is related to cognitive and functional decline. In addition, donepezil treatment induced the decrease of plasma EVs levels of t-tau, P-T181-tau and P-S396-tau in patients with mild to moderate AD.