Abstract: In order to determine the optimal conditions of shaking flasks fermentation of Saccharomy cescerevisiae, we conducted the single- factor test, including culture temperature, culture time and rotation speed. The L₉(3³) orthogonal test was used to optimize the culture conditions of Saccharomy cescerevisiae, and the L9(34) orthogonal test was used to optimize the fermentation culture media of Saccharomy cescerevisiae. The results showed that, the optimization fermentation condition was culturing in 29℃, speed 160 r/min for 27 hours. The optimization fermentation culture media was glucose 20 g/L, yeast powder 10 g/L，KH₂PO₄ 1.5 g/L，MgSO₄ 1 g/L. The key factors affecting the growth of Saccharomyces cerevisiae were the culture time and the glucose concentration of the culture medium.