In ethanol fermentation, by-products such as glycerol, acetic acid and lactic acid are produced along with ethanol. The effects of culture conditions on cell growth ethanol production and by-products biosynthesis were investigated in ethanol fermentation using S. cerevisiae. With increasing aeration rate or yeast extract concentration, ethanol and by-products biosynthesis decreased while final cell mass increased. With increasing glucose concentration or decreasing temperature, final cell mass, ethanol and by-products concentrations all increased. The optimal pH for the cell growth, ethanol and by-products productions was found to be pH 4.5. By-products biosynthesis was found, in general, to proceed with the ethanol biosynthesis. The results can be applied for the optimization of ethanol fermentation and for the recovery and purification of ethanol from the culture broth.