Abstract: Xylose can be oxidated to xylitol by xylosere duetase, which the reaction is the node position in the xylose metabolism. Xylose can’t be utilized by Saccharomyces cerevisiae because of its lack of xylosere duetase. Therefore, the xyl1 should be inserted into the genome of Saccharomyces cerevisiae in order to consummate xylose metabolic flux, and enhance xylose utilization rate as well as the ethanol yield by Saccharomyces cerevisiae. In this paper, according to the sequence similarity of xylose reductase, polymerase chain reaction (PCR) was used to amplify the xyl1gene, a pair of primers were designed considering the Candida shehatae genome DNA and plasmid PKT0150 as template and finally 1110 bp cloning xyl1of xylose reductase gene was obtained with three base missing, The ORF located in 11-982, total length of 972 bp, encoding 323 amino acids after missing three bases TTG in the 10, 11, 12 of ORF. Results showed that the segment was the xylose reductase gene sequencesas in HDYXHT-20335 which laid the foundation for the construction of the yeast strain.