The invention provides application of trilobatin in preparation of a medicine for preventing and/or treating non-alcoholic fatty liver disease. The trilobatin is researched and found for the first time to be capable of improving blood glucose metabolism disorder, abnormal blood lipid metabolism, liver function impairment, liver inflammatory response, liver injury, liver fatty degeneration and lipid deposition caused by the non-alcoholic fatty liver disease, and the trilobatin and the derivative thereof have a good treatment effect on the non-alcoholic fatty liver disease. The invention provides a novel and efficient natural medicine source for treating the non-alcoholic fatty liver disease.