β-Galactosidase production by Bifidobacterium longum CCRC 15708, Bifidobacterium longum B6 and Bifidobacterium infantis CCRC 14633 was first examined with B. longum CCRC 15708 showing the highest production of β-galactosidase and the highest specific activity. Further study with B. longum CCRC 15708 revealed that the highest level of β-galactosidase was produced with lactose and yeast extract as carbon and nitrogen sources, respectively. Optimal enzyme production occurred at an initial pH of 6.5 and at 37 °C. Under these optimum culture conditions, a maximumβ-galactosidase activity of 18.6 U/ml could be obtained after 16 h of fermentation in a medium contain 4% lactose, 3.5% yeast extract, 0.3% K 2 HPO 4 , 0.1% KH 2 PO 4 , 0.05% MgSO 4 ·7H 2 O and 0.03% l-cysteine. The highest transgalactosylation activity was also detected in this culture after 14–16 h of fermentation.