Fermentation properties under the control of multiple genes of industrial Saccharomyces cerevisiae strain are difficult to alter with traditional methods. Here, we describe efficient and reliable genome shuffling to increase ethanol production through the rapid improvement of stress resistance. The strategy is carried out using yeast sexual and asexual reproduction by itself instead of polyethylene glycol-mediated protoplast fusion. After three rounds of genome shuffling, the best performing strain S3-10 was obtained on the special plate containing a high ethanol concentration. It exhibits substantial improvement in multiple stress tolerance to ethanol, glucose, and heat. The cycle of fermentation of S3-10 was not only shortened, but also, ethanol yield was increased by up to 10.96% compared with the control in very-high-gravity (VHG) fermentations. In total, S3-10 possesses optimized fermentation characteristics, which will be propitious to the development of bioethanol fermentation industry.