Expression of miR-28-3p in patients with Alzheimer's disease (AD) before and after treatment and clinical value of miR-28-3p were determined. There were three groups: 68 AD patients treated with donepezil combined with basic therapy in The People's Hospital of Shouguang collected as an AD group, 70 patients with mild cognitive impairment (MCI) as an MCI group, and 75 healthy people as a normal group. Serum miR-28-3p was detected by qRT-PCR. The Montreal cognitive assessment scale (MoCA), mini mental state examination scale (MMSE), activities of daily living scale (ADL) and homocysteine (Hcy) were adopted to assess patients before and after treatment. miR-28-3p in normal group was significantly lower than that in other two groups, and miR-28-3p in MCI group was significantly lower than that in AD group (P<0.001). miR-28-3p correlated with the course and severity of patients. miR-28-3p in AD group after treatment was significantly lower than that before treatment (P<0.001). ADL and Hcy of AD patients after treatment were significantly lower than before treatment (P<0.05), and MMSE and MoCA after treatment were significantly higher than before treatment (P<0.05). Before and after treatment, miR-28-3p was significantly positively correlated with ADL score and Hcy level, but negatively correlated with MMSE score and MoCA score. Analysis of the working characteristic curve of the patients indicated that miR-28-3p can be used for diagnosis of AD patients. Donepezil therapy may reduce miR-28-3p level to alleviate the symptoms of AD patients, and miR-28-3p level can be used as an early diagnosis and prognosis evaluation of AD patients.