Utilization of lignocellulose materials to replace the pure glucose for preparation of the fermenting yeast seeds could reduce the cost of ethanol fermentation, because a large quantity of glucose is saved in the large-scale seed fermentor series. In this study, Saccharomyces cerevisiae DQ1 was cultured using the freshly pretreated corn stover material as the carbon source, and then the culture broth was used as the inoculation seeds after a series of seed transfer and inoculated into the ethanol production fermentor. The results show that the yeast cell growth and ethanol fermentation performance have essentially no difference when the yeast seeds were cultured by glucose, the corn stover hydrolysate liquid, and the pretreated corn stover solids as carbon sources, respectively. Approximately 22% of the yeast cell culture cost was saved, and the process flow sheet in industrial scale plants was simplified by using the pretreated corn stover for seed culture. The results provided a practical method for materials and operational cost reduction for cellulosic ethanol production.