The xylulokinase gene XKS1 was cloned from Saccharomyces cerevisiae NAN-27 and ligated into plasmids pMA91 and YEp24, producing pMA-xy203 and YEpP-xy204, respectively. In both plasmids, XKS1 was under the control of PGK promoter. pMA-xy203 was transferred into the pre-constructed recombinant yeast strain H158-XR-XDH, which contains the XYL1 and XYL2 genes from Pichia stipitis, encoding xylose reductase and xylitol dehydrogenase respectively, in an episomal plasmid vector. This new recombinant strain was named HSXY-251. YEpP-xy204 was transferred into the pre-constructed recombinant yeast strain H158-XI, which contains the xylA gene from Thermus thermophilus encoding xylose isomerase in an episomal plasmid vector, resulting in recombinant strain HSXY-252. The xylulokinase activities in HSXY-251 and HSXY-252 were respecfively 14 and 6.7 times higher than that in the parent strain. Glucose and xylose co-fermentation carried out with HSXY-251 under oxygen-limited conditions at 30℃ resulted in 9.4 g/L ethanol concentration with 12.4 g/L xylose consumed. Xylose consumption and ethanol production were respectively 120.9% and 36% higher than in the parent strain. Furthermore, the by-product xylitol was 0.7 g/L, a decrease of 84.9%.