The invention provides tobacco flavor fermented by utilizing radix ophiopogonis and a preparation method and application thereof and belongs to the field of the tobacco flavor and the preparation method and application thereof. The preparation method comprises the steps of drying and smashing radix ophiopogonis, using flavor-producing yeasts to ferment and add flavor, then using an organic solvent to perform extraction, and performing solid-liquid separation and concentration to prepare the tobacco flavor. A finished tobacco flavor fermented by utilizing the radix ophiopogonis is diluted with ethanol or propylene glycol and then evenly sprayed on tobacco shreds according to 0.01%-0.1% of tobacco weight. The tobacco flavor fermented by utilizing the radix ophiopogonis has the advantages that microbial fermentation biotransformation process is adopted, so that the tobacco flavor fermented by utilizing the radix ophiopogonis is full and velvety compared with radix ophiopogonis flavor prepared by means a conventional method, flavor-producing yeast fermentation enables the radix ophiopogonis to produce aroma components harmonious with cigarettes, ester flavor is added and harmonious with the tobacco flavor except sweet flavor of the radix ophiopogonis, stimulation is reduced, and offensive odor is decreased.