The cost effective production of ethanol from lignocellulose plant biomass requires the fermentation of all available sugars. Xylose the second abundant sugar in plant biomass could not be fermented by Saccharomyces cerevisiae. In this study, a recombinant S. cerevisiae was constructed to utilise xylose and ferment it to ethanol. This was achieved by isolating the genomic DNA from Spathaspora passalidarum and used as a template for xylose reductase (XYL1) and xylitol dehydrogenase (XYL2) genes amplification by Polymerase chain reaction (PCR). The two amplified genes were cloned into a shuttle vector (pSN303 plasmid) to obtain two plasmids designated as pYIM1 and pYIM2 respectively. These two plasmids were used to transform Escherichia coli and the obtained strains were designated as YJTE1 and YJTE2 respectively. The presence of these plasmids were confirmed using SacII and NotI restriction endonucleases and PCR. The correct Open reading frame (ORF) for these two genes within the plasmids were confirmed by sequencing. The two plasmids were isolated from E. coli and used to transform Saccharomyces cerevisiae. Transformants carrying the two plasmids were designated as YJTY1 and YJTY2 respectively. These strains were grown on SCD–URA- medium and the cells were broken to assay Xylose reductase (XR) and Xylitol dehydrogenase (XDH) specific activities. Xylose reductase enzyme could convert xylose to xylitol with specific activities of 2.41 U mg-1 protein and 1.17 U mg-1 protein on both NADH and NADPH cofactors respectively. Xylitol dehydrogenase enzyme showed a high specific activity 22.81 U mg-1 protein converting xylitol to xylulose with NAD+ cofactor whereas it did not show any specific activity on NADP+ cofactor. Then, full pathway of xylose utilisation with enhanced efficiency were constructed by the following: the pYIM1 plasmid was digested with NotI then XYL2 and xylulokinase (XYL3) genes from S. passalidarum and ransaldolase (TAL1) from S. cerevisiae were amplified using PCR. Furthermore, gene promoters of Transcription elongation factor (TEF1), Phosphoglycerate kinase (PGK1), Alcohol dehydrogenase (ADH1) and gene terminators including PGK1, TEF1, and Cytochrome C (CYC1) from S. cerevisiae were also amplified using PCR. All the above DNA fragments including the digested plasmid were assembled using Gibson method to obtain pYIM3 plasmid. This plasmid was used to transform the E. coli and the resultant strain was designated as YJTE3. The presence of this plasmid with all the DNA fragments was confirmed using PvuI and PvuII restriction endonucleases and the correct nucleotide sequence was confirmed by sequencing. This plasmid was isolated, used to transform S. cerevisiae and the resultant strain was designated as YJTY3. This strain was compared with the control strain YJTY0 carrying the pSN303 plasmid for xylose fermentation. Several fermentation experiments were done under oxygen limited conditions. After three subcultures, the results were as follow: specific xylose consumption rate 0.103 g g-1 DCW h-1, specific growth rate 0.025 h-1, specific ethanol production rate 0.046 g g-1 DCW h-1, and specific xylitol production rate 0.0016 g g-1 DCW h-1. Furthermore, ethanol yield was 0.447 g g-1 of consumed sugar which was about 90 % of the theoretical yield 0.51 g g-1. These results showed that the recombinant strain YJTY3 obtained from this study was able to utilise xylose and produce ethanol.