Lactose was introduced into soybean protein isolate (SPI) via glycosylation to produce an SPI-lactose conjugates, under different temperatures, protein and sugar mass ratios, as well as reaction durations. Subsequent changes in the antigenicity of β-conglycinin in the SPI-lactose conjugates was estimated by indirect-competition enzyme-linked immunosorbent assay (ic-ELISA). The structural properties of the SPI-lactose conjugates were also studied. The results indicated that glycosylation reduced the antigenicity of β-conglycinin from 93.79% (control) to 37.75% (conjugates). Compared to SPI, the content of free amino groups in the complex also decreased after glycosylation. The reduction was highest at 60 hours after the of the reaction. Fourier transformed infrared spectroscopy (FT-IR) showed that the quantity of α-helices, β-turns, and random coil structures were lower in the SPI-lactose glycosylation product than that in the SPI, while that of β-sheet increased. Results of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and periodic acid-Schiff (PAS) staining showed that the SPI band gradually became weaker with the progress in glycosylation reaction, indicating that the glycosylation reaction occurred between SPI and lactose molecules.