High ethanol tolerance is a desired property of industrial yeast strains for efficient ethanol fermentation. In this study, the impact of medium composition on ethanol tolerance of the self-flocculating yeast SPSC01 was investigated using a chemically defined medium. Singlefactor experiments revealed that besides magnesium and calcium, zinc also exhibited significant protective effect against ethanol toxicity; addition of 0.02 g/l zinc sulfate significantly increased cell viability in the ethanol shock treatment. Metal ions of manganese, cobalt, and ferrous failed to promote ethanol tolerance, although addition of 0.02 g/l cobalt increased ethanol production without apparent influence on ethanol tolerance. Furthermore, Uniform Design method was employed to obtain the medium with high cell viability, and the key nutrient factors in the medium composition were revealed to be (NH4)2SO4, K2HPO4, vitamin mixtures, and the metal ions of magnesium, calcium and zinc. The optimized combination of metal ions addition was (g/l): MgSO4 0.4, CaCl2 0.2, ZnSO4 0.01. The highest cell viability (90.2%) of SPSC01 against ethanol shock treatment was observed in the optimized medium, which demonstrated significant improvement of ethanol tolerance of the self-flocculating yeast.