Abstract: In order to establish soybean globulin allergen detection method and epitope mapping of protein allergenic subunit, soybean globulin was isolated and purified from defatted soybean powder to prepare polyclonal antiserum, and its immunological characteristics were analyzed. Soybean globulin isoelectric point pH is 6.4. Soybean globulin was crudely extracted by alkali extraction and acid precipitation, followed by purified by Sepharose CL-6B chromatography and its purity was detected by SDS-PAGE. New Zealand rabbits were immunized with soybean globulin to prepare polyclonal antiserum, and its immunological characteristics were analyzed. The results showed that the titer of rabbit No.1 was 1∶6 ×10⁴, and the half inhibition rate IC₅₀ was 527 ng/mL, which showed the highest sensitivity. The specificity results demonstrated that the cross-reaction rate of the serum with protein was less than 0.2%, except for β-conglycinin and peanut protein (8.6% and 4.6%.) The prepared rabbit polyclonal antiserum had high titer, sensitivity and specificity, which layed a foundation for the preparation of soybean globulin monoclonal antibody and the mapping of soybean protein allergenic submolecular structure.