Abstract: In this study, the glucose oxidase gene with five amino acid sites-directed mutagenesis was synthesized.The activity of this mutant enzyme was 3.24 times than that of the wild type enzyme. The episomal vector p KDU7 and integrative vector p KLAC1 in which the glucose oxidas gene was recombined was constructed. Using these two vectors, the glucose oxidase gene was successfully recombined into K. lactis Kl SEL1 strain and a recombinant strain with efficient secretory over-expression of glucose oxidase was obtained.The result of flask-shaking fermentation showed that the production of glucose oxidase reached 70± 7 k U/L in the recombinant strain.This is the highest glucose oxidase-producing recombinant K.lactis strain reported so far.This technology will provide a promising approach for the production and application of glucose oxidase in food industry.