Abstract: This study aimed to provide a new lactic acid bacteria (LAB) enzyme source for synthesizing probiotic galactooligosaccharides. It screened β-galactosidase producing strains with high transglycosidase activity from traditional cheese in Xinjiang, China. A modified MRS medium with lactose as the single carbon source supplemented with 5-bromo-4-chloro-3-indole-β-D-galactoside was used for the first-round screening of LAB strains producing β-galactosidases. Thin layer chromatography (TLC) analysis of the reaction products catalyzed by the crude enzyme from selected strains was used for the second-round screening. The isolate with the highest transglycosylation activity was identified based on the physiological and biochemical characteristics, along with the sequence analysis of the 16S rRNA gene. The conditions for enzyme production and transglycosylation reaction were determined by one-way experiment. TLC combined with high performance liquid chromatography was conducted to analyze the content of each GOS component. Six strains producing β-galactosidase with transglycosidase activity were screened, among which Pediococcus acidilactici Y1 exhibited the highest enzyme production level and transglycosidase activity. After optimization, P. acidilactici Y1 produced β-galactosidase at a level of up to 15.52±0.34 U/g (with 20 mg/mL of initial lactose after 18 h at 37 ℃). The highest GOS yield of 38.4%±0.56% (w/w) was achieved with an initial lactose concentration of 300 mg/mL, an enzyme amount of 4.24 U/mL, and a reaction temperature of 50 ℃ for 28 h. Also, the residual rate of lactose decreased to 30.9%±0.44%(w/w). The content of transfer disaccharide, transfer trisaccharide, and transfer trisaccharide was 15.6%±0.21% (w/w), 18.3%±0.15% (w/w), and 4.5%±0.01% (w/w), respectively. The results of this study showed that P. acidilactici Y1 was a new β-galactosidase-producing strain, with application prospects in the synthesis of probiotic GOS.