β-glycosidase from bovine liver is a biocatalyst that can catalyze the synthesis of disaccharide nucleosides via regioselective glycosylation of nucleosides. In previous reports, the used biocatalysts were free enzymes, which limit the large-scale applications. As novel carrier-free immobilized enzymes, cross-linked enzyme aggregates (CLEAs) have many advantages, such as easy preparation, low cost, and high enzyme volumetric activities. CLEAs of β-glycosidase from bovine liver were prepared, their enzymatic properties and structures were studied, and their application for the synthesis of disaccharide nucleoside was explored. The optimal precipitant, cross-linking agent and cross-linking time were 70% saturated ammonium sulfate, 50% (V/V) dextran polyaldehyde, and 8 h, respectively. Under optimal conditions, the activity recovery of CLEAs reached 58%. The optimal pH, temperature, Vmax and Km of the CLEAs were 8.5, 60 ℃, 0.13 μmol/(min?mg), and 1.43 mM, respectively. Additionally, the CLEAs showed good operational stability. After they were reused for six batches, the relative yield in the CLEA-catalyzed synthesis of 5′-O-β-glucosyl-2′-deoxyuridine had decreased only by 20%. The CLEAs had an amorphous structure.