To prepare monoclonal antibody（mAb） against Bowman-Birk inhibitor（BBI） with good immunological characteristics， BALB/c mice were used to immunize with 50 μg of BBI， and the antiserum titer and sensitivity were identified via indirect ELISA and indirect competitive ELISA. The mice which antiserum has better titer and sensitivity was selected for cell fusing through cell hybridoma technique， stable hybridoma cell lines that could secrete BBI mAb were obtained via multiple screening， and the BBI mAb was prepared by inducing ascitic fluids in vivo and identified the immunological characteristics. The result indicated that the NO.1 mouse was confirmed to produce the antibody with the highest titer and sensitivity， which half inhibitory concentration（IC50） was 868.58 ng/mL. After cell fusing， five hybridoma cell strains were screened. The 3B7-B10 mAb titer was above 1 ∶ 4.096×105. Its subtype was IgG1 and IC50 was 83.07 ng/mL. It had high affinity and specificity， demonstrated the mAb had good immunological characteristics. This study had provided the favorable antibody basis for the establishment of immunological detection of BBI in soybean and its products.