<P>Xylanase is a widely-used additive in baking industry for enhancing dough and bread quality. Several xylanases used in baking industry were expressed in different systems, but their expression in antibiotic free vector system is highly essential and safe. In the present study, an alternative rDNA-mediated technology was developed to increase the copy number of target gene by integrating it into <I>Saccharomyces cerevisiae</I> genome. A xylanase-encoding gene <I>xynHB</I> from <I>Bacillus sp</I>. was cloned into pHBM367H and integrated into <I>S. cerevisiae</I> genome through rDNA-mediated recombination. Exogenous XynHB expressed by recombinant <I>S. cerevisiae</I> strain A13 exhibited higher degradation activity towards xylan than other transformants. The real-time PCR analysis on A13 genome revealed the presence of 13.64 copies of <I>xynHB</I> gene. Though no antibiotics have been used, the genetic stability and the xylanase activity of <I>xynHB</I> remained stable up to 1,011 generations of cultivation. <I>S. cerevisiae</I> strain A13 expressing xylanase reduced the required kneading time and increased the height and diameter of the dough size, which would be safe and effective in baking industry as no antibiotics-resistance risk. The new effective rDNA-mediated technology without using antibiotics here provides a way to clone other food related industrial enzymes for applications.</P>