A method of preparing a yeast usable in a fermentation test for evaluating malt on the laboratory scale whereby more reproducible test data can be obtained regardless of the operation conditions of a plant, etc.; and a medium to be used therein. A yeast for the fermentation test for evaluating malt is prepared by culturing a yeast in a liquid medium containing 0 to 2.5% by weight of glucose, 6.0 to 9.0% by weight of maltose, 0 to 2.5% by weight of sorbitol, 0 to 1.0% by weight of peptone and 0.3 to 1.0% by weight of yeast extract at 10 to 20.degree. C. for 2 to 4 days under shaking (primary culture), then in the medium in an increased amount compared with the primary culture at 10 to 20.degree. C. for 3 or 4 days under shaking (secondary culture), then in the medium in a further increased amount compared with the secondary culture at 8 to 9.degree. C. for 7 to 9 days under stirring until the plats attains 3.0.degree. P. or less (tertiary culture), allowing to stand in a cold room for 3 to 24 hours and then collecting the yeast.