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Expression and purification of β-galactosidases derived from <i>Bifidobacterium</i> provide a new resource for efficient lactose hydrolysis and lactose intolerance alleviation. Here, we cloned and expressed two β-galactosidases derived from <i>Bifidobacterium</i>. The optimal pH for BLGLB1 was 5.5, and the optimal temperature was 45 °C, at which the enzyme activity of BLGLB1 was higher than that of commercial enzyme E (300 ± 3.6 U/mg) under its optimal conditions, reaching 2200 ± 15 U/mg. The optimal pH and temperature for BPGLB1 were 6.0 and 45 °C, respectively, and the enzyme activity (0.58 ± 0.03 U/mg) under optimum conditions was significantly lower than that of BLGLB1. The structures of the two β-galactosidase were similar, with all known key sites conserved. When o-nitrophenyl-β-D-galactoside (oNPG) was used as an enzyme reaction substrate, the maximum reaction velocity (<i>V</i>_max) for BLGLB1 and BPGLB1 was 3700 ± 100 U/mg and 1.1 ± 0.1 U/mg, respectively. The kinetic constant (<i>K</i>_m) of BLGLB1 and BPGLB1 was 1.9 ± 0.1 and 1.3 ± 0.3 mmol/L, respectively. The respective catalytic constant (<i>k</i>_cat) of BLGLB1 and BPGLB1 was 1700 ± 40 s⁻¹ and 0.5 ± 0.02 s⁻¹, respectively; the respective <i>k</i>_cat/<i>K</i>_m value of BLGLB1 and BPGLB1 was 870 L/(mmol∙s) and 0.36 L/(mmol∙s), respectively. The <i>K</i>_m, <i>k</i>_cat and <i>V</i>_max values of BLGLB1 were superior to those of earlier reported β-galactosidase derived from <i>Bifidobacterium</i>. Overall, BLGLB1 has potential application in the food industry.