Active peptides or food enzymes with natural antioxidant function are green， safe and reliable， and have attracted wide interest as green and multifunctional food additives. Hexose oxidase (HOX)， a D-hexose:O₂ 1-oxidoreductase， and has the functions of deoxygenation， acidification and improving protein crosslinking. In order to optimize the expression strategies of hexose oxidase in Pichia pastoris， different genotype host strains， different gene expression modes and different gene copy number in transformants were systematically compared. The transformants with different genotypes were obtained by the newly established three-step screening method， and their enzyme activities by fermentation were compared. The results showed that Muts host with slow methanol metabolism generated higher HOX activity； inducible expression by methanol was better than constitutive expression by GAP promoter； higher gene copy number of HOX gene generated higher enzyme activity. Although these factors related to gene expression in P. pastoris had been optimized， the HOX activity was still in a low level， and the maximum activity produced by KCX56 strain was 4.92×10-2 U/mL， which contained six gene copy number. The purification of recombinant HOX showed that there were three recombinant proteins with the molecular weight of 62， 40 ku and 29 ku， respectively. They should be protoenzyme of hexose oxidase and two subunits formed by proteolytic cleavage. Among them， the proportion of protoenzyme with low activity was much higher than other two， which may be one of the reasons for the low fermentation activity by these recombinant strains. The study of enzymatic characteristics showed that the optimum conditions for the recombinant HOX were 50-60 ℃ and pH 4.0， and it can maintain 50% activity at room temperature (20 ℃). Under low temperature (20-60 ℃) and acidic conditions (pH 3.0-6.0)， HOX activity can be stably maintained， so it is an antioxidant enzyme with good acid resistance， meaning that HOX is suitable for the preservation and antioxidant application for acidic foods.