The invention relates to the technical field of spreading cultivation of microbial strains and fermentation of ethanol and discloses a method for spreading cultivation of saccharomycetes by virtue of liquefied mash, application of the method and a method for fermenting ethanol. The method for spreading cultivation of saccharomycetes by virtue of liquefied mash comprises the step of carrying out intermittent spreading cultivation and fed-batch spreading cultivation on activated saccharomycetes by utilizing liquefied mash filtrate under an aseptic condition. According to the method, the number of the saccharomycetes can be effectively controlled, the obtained saccharomycetes have relatively high activities, and the yield of ethanol and the consumption rate of xylose in a fermentation process can be obviously increased.