Lactobacillus acidophilus is a kind of probiotics. The previous research found that β-glucosidase (BGL) produced by Lactobacillus acidophilus GIM1.208 has high activity. In order to elucidate its structure and characteristics, the target gene of Lactobacillus acidophilus GIM1.208 BGL was obtained with PCR and successfully expressed in E. coli. Protein samples with a purity of more than 90% was obtained by nickel affinity chromatography. Then the secondary structure of BGL was detected by circular dichroism spectroscopy (CD) and its tertiary structure was analyzed by homology modeling method. The enzymatic properties of Lactobacillus acidophilus BGL was also studied. The results show that the molecular mass of Lactobacillus acidophilus BGL is 52 ku, the concentration after purification is 1.88 g/L. The secondary structure of Lactobacillus acidophilus BGL includes 15.9% α-helix, 44.1% β-sheet, 18.1% β-turn, and 27.2% random coil. Homology modeling analyzation showed that 8 β-sheets and 8 α-helices were included in 3D structure of Lactobacillus acidophilus BGL, and the whole protein is conical in shape. Lactobacillus acidophilus BGL has good glucose tolerance and NaCl tolerance. The optimal temperature and the optimal pH of this enzyme is 47℃ and 5.6, respectively. Lactobacillus acidophilus BGL has high stability in the range of 20℃-50℃ and pH 2.2-6.0. The inhibitory effect of ethyl acetate and methanol on enzyme activity is obvious, and Fe³⁺ and Fe²⁺ have significant activation effect on enzyme. These results provide important reference basis for the subsequent functional exploration and application research of Lactobacillus acidophilus BGL.