The efficient fermentation of xylose can improve biofuel production. We previously developed a two-stage transcriptional reprogramming (TSTR) strategy (including a glucose fermentation stage and a xylose fermentation stage) and demonstrated its application for the construction of Saccharomyces cerevisiae strains with efficient xylose utilization. In this study, we used these as initial strains to assess the effects of copy number variation (CNV) on optimal gene expression and rewiring the redox balance of the xylose utilization pathway. We obtained strains that contained several integrated copies of XYL1, XYL2, and XKS1 and showed increased ethanol yields. An examination of the individual and combined effects of CNVs of key genes and the redox balance pathway revealed that the TSTR strategy improves ethanol production efficiency. Furthermore, XYL1 or XYL2 overexpression was related to improved xylose utilization. These results showed that strains with faster growth and/or higher ethanol production produced more ethanol from xylose via the synthetic xylose-assimilation pathway. Accordingly, TSTR is an effective strategy to improve xylose metabolism in industrial yeast strains.