Abstract: Objective: To analyze a novel cold-adapted β-galactosidase, which provided the basis for producing low lactose dairy products or synthesizing galactooligosaccharides (GOS) at low temperatures. Methods: The β-galactosidase (PpBgal42A) gene was cloned from the genome of Paenibacillus polymyxa. The recombinant plasmid (pET-28a-PpBgal42A) was constructed and successfully expressed in E.coli BL21(DE3). After purification by affinity chromatography, the enzymatic properties of recombinant β-galactosidase were studied. To evaluate the transglycosylation ability of PpBgal42A, the concentration of each component was determined by high performance liquid chromatography with the conversion rate of GOS as evaluation index. Results: PpBgal42A shared the highest identity of 59.9% with the GH family 42 β-galactosidase from Alicyclobacillus acidocaldarius. After purification, the specific activity of PpBgal42A was 163.7 U/mg and the molecular weight was about 79 kDa. Its optimal pH and temperature were pH7.5 and 35 ℃, respectively. It was stable in the range of pH7.0~9.5 and 4~35 ℃. It was rapidly inactivated at 50 ℃. The half-life of PpBgal42A at 35 ℃ was 1777 min. PpBgal42A synthesized 31.6% of GOS using 350 g/L lactose as the substrate at 30 ℃ for 6 h. Conclusion: PpBgal42A is a novel cold-adapted enzyme. It can hydrolyze lactose and synthesize galactooligosaccharides, which has potential application value.