The invention discloses application of isosteviol to preparation of a medicament for treating non-alcoholic fatty liver disease. Compared with the prior art, the invention discovers that the isosteviol can improve rat non-alcoholic fatty liver disease caused by high fat diet, reduce the accumulation of lipid in hepatocytes, restrain liver intracellular oxidative stress, impaired mitochondrial function and liver apoptosis caused by free fatty acid, and protect liver cells. On the aspect of overall animal level, the serum transaminase level is lowered, the free fatty acid in serum is reduced, and dyslipidemia is improved. The functions indicate that the isosteviol can be applied to treatment of the non-alcoholic fatty liver disease, and has a medicament development prospect.