We screened and identified a set of efficient promoters in Saccharomyces cerevisiae that maintained their relatively strong strengths to regulate the heterologous xylose-assimilating pathway genes XYL1 and XYL2, and native XKS1 and pentose phosphate pathway four genes, irrespective of glucose or xylose fermentation medium. In this study, we developed a rapid and efficient xylose-fermenting S. cerevisiae strain 7-1 based on balanced pathway expression levels driven by our proposed strong promoters. Next, 7-1 was used to initialize the evolutionary engineering, through first aerobic and anaerobic sequential batch cultivation. The finally evolved strain of 7-1E1 displayed a high ethanol yield (0.45 g/g) and low xylitol accumulation (0.13 g/g). Moreover, the evolved strain of 7-1E1 displays great potential for ethanol production from lignocellulosic biomass. This work reveals that efficient xylose assimilation is attributed to the elevated expression levels of xylose utilization genes, which was accomplished through the strong promoter rational regulation in the chromosome of the evolved strain.