In this study, we utilized a unique strategy for fed-batch fermentation using ethanol-tolerant Saccharomyces cerevisiae to achieve a high-level of ethanol production that could be practically applied on an industrial scale. During this study, the aeration rate was controlled at 0.0, 0.13, 0.33, and 0.8 vvm to determine the optimal aeration conditions for the production of ethanol. Additionally, non-sterile glucose powder was fed during fed-batch ethanol fermentation and corn-steep liquor (CSL) in the medium was used as an organic N-source. When aeration was conducted, the ethanol production and productivity were superior to that when aeration was not conducted. Specifically, the maximum ethanol production reached approximately 160 g/L, when the fermentor was aerated at 0.13 vvm. These findings indicate that the use of a much less expensive C-source may enable the fermentation process to be directed towards the improvement of overall ethanol production and productivity in fermentors that are aerated at 0.13 vvm. Furthermore, if a repeated fed-batch process in which the withdrawal and fill is conducted prior to 36 h can be employed, aeration at a rate of 0.33 and/or 0.8 vvm may improve the overall ethanol productivity.