A beta-galactosidase gene from Aspergillus oryzae was engineered utilizing codon usage optimization to be constitutively and highly expressed in the Pichia pastoris SMD1168H strain in a high-cell-density fermentation. After fermentation for 96 h in a 50-L fermentor using glucose and glycerol as combined carbon sources, the recombinant enzyme in the culture supernatant had an activity of 4,239.07 U mL(-1) with o-nitrophenyl-beta-D-galactopyranoside as the substrate, and produced a total of extracellular protein content of 7.267 g L-1 in which the target protein (6.24 g L-1) occupied approximately 86 %. The recombinant beta-galactosidase exhibited an excellent lactose hydrolysis ability. With 1,000 U of the enzyme in 100 mL milk, 92.44 % lactose was degraded within 24 h at 60 degrees C, and the enzyme could also accomplish the hydrolysis at low temperatures of 37, 25, and 10 degrees C. Thus, this engineered strain had significantly higher fermentation level of A. oryzae lactase than that before optimization and the beta-galactosidase may have a good application potential in whey and milk industries.