Galactooligosaccharides are important prebiotics. β-Galactosidases from Bacillales sp. can catalyze the transfer of galactoside, which can be used for the synthesis of galactooligosaccharides. The expression, enzymatic properties and application in the synthesis of galactooligosaccharides of a novel β-galactosidase from Bacillales sp. were investigated. A glycoside hydrolase (GH) family 35 β-galactosidase gene (BABgal35A) from Bacillales sp. was cloned and expressed in E. coli BL21(DE3). The results of multiple sequence alignment showed that BABgal35A shared the highest identity of 79.9% with a β-galactosidase from Bacillus circulans, and was a novel β-galactosidase (named BABgal35A). Crude enzyme was purified through Ni-IDA affinity chromatography to obtain electrophpresis-grade pure enzyme. SDS-PAGE showed that the molecular weight of purified enzyme was about 60kDa. The optimal pH and temperature of BABgal35A were determined to be 5.0 and 55℃, respectively. The enzyme was stable at pH 4.5-8.0 and temperature 20-45℃. BABgal35A exhibited the highest specific activity towards oNP-β-galactopyranoside. A maximum yield of 34% for galactooligosaccharides production was obtained by BABgal35A using lactose as substrate. The study showed that a GH family 35 β-galactosidase from Bacillales sp. was potentially suitable for the preparation of galactooligosaccharides.