The invention discloses a method for preparing a yeast culture through distillers' grains and an application of the yeast culture. The method comprises the following steps of S1, supplementing a carbon source to distillers' grains to obtain pretreated distillers' grains, inoculating mixed bacterium liquid to the pretreated distillers' grains, adding mixed enzyme liquid, preparing a fermentation base material, and performing fermentation culture for 96-120h; and S2, after fermentation is completed, performing inoculation with bacillus subtilis, continuing to perform fermentation culture for 24h, and performing baking, crushing and screening so as to obtain the yeast culture, wherein the mixed bacterium liquid comprises 3 strains of saccharomyces cerevisia, lactic acid bacteria and aspergillus niger, and the mixed enzyme liquid comprises liquefying amylase, glucoamylase, phytase and proteases. According to the method disclosed by the invention, the distillers' grains are used as substrates, the mixed bacterium liquid and the mixed enzyme liquid are added for heap fermentation, so that the yeast culture is obtained; and the distillers' grains are turned into wealth from waste, and besides, a feed rich in nutrient components is provided for animals, so that improvement of the immunity of the animals can be facilitated.