The invention relates to a medicine for preventing and treating diabetes mellitus based on hsa-miR-320a, and a screening method and a preparation method thereof, and belongs to the fields of molecularmedicine, and diagnosis, prevention and treatment of metabolic diseases. The hsa-miR-320a is adopted as a target of the medicinal component of the medicine, and effects of preventing and treating diabetes mellitus are achieved through combining, and/or capturing, and/or degrading, and/or down-regulating expression of the hsa-miR-320a. The hsa-miR-320a is shown as SEQ ID NO.1. The screening methodand the preparation method of the medicine are also provided. The medicine can significantly reduce the blood glucose concentration of a patient with diabetes mellitus, can effectively relieve diabetic complication symptoms of animal subjects, and can allow animal subjects to maintain healthy and stable blood glucose levels in long time.