A gene of glucose oxidase (GOD) from Aspergillus niger Z-25 was cloned and sequenced. The entire open reading frame (ORF) consisted of 1,818 bp and encoded a putative peptide of 605 amino acids. The gene was fused to the pPICZ alpha A plasmid and overexpressed in Pichia pastoris SMD1168. The recombinant GOD (rGOD) was secreted into the culture using MF-alpha factor signal peptide under the control of the AOX1 promoter. Sodium dodecyl sulfate polyacrylamide gel electrophoresis indicated that rGOD exhibited a single band at around 94 kDa. The maximal GOD activity of approximately 40 U/mL was achieved in shake flask by induction under optimal conditions after 7 days. rGOD was purified by ammonium sulfate precipitate leading to a final specific activity of 153.46 U/mg. The optimum temperature and pH of the purified enzyme were 40 A degrees C and 6.0, respectively. Over 88% of maximum activity was maintained below 40 A degrees C. And the recombinant enzyme displayed a favorable stability in the pH range from 4.0 to 8.0. The Lineweaver-Burk plotting revealed that rGOD exhibited a K (m) value of 16.95 mM and a K (cat) value of 484.26 s(-1).