To prepare monoclonal antibodies against soybean Kunitz trypsin inhibitor (KTI) with good immunological characteristics. BALB/c mice were used to immunize with 50 μg of KTI, the antiserum titer and sensitivity were identified via indirect ELISA and indirect competitive ELISA. The mice which serum has better titer and sensitivity were selected for cell fusing, then he hybridoma cell strain which stabilization secreted KTI monoclonal antibody (mAb) were screened. Thereafter, mAb was prepared by inducing ascites in vivo and identified the immunological characteristics. The result indicated that the NO.1 mouse was confirmed to produce the antibody with the highest titre, IC₅₀ was 157.33 ng/mL. After cell fusing, 4E6-E9 positive hybrid tumor cell lines were obtained, and the 4E6-E9 mAb titer was above 11 638 400, of which subtype was IgG1, with affinity constant 1.45×10⁸ L/mol, IC₅₀ 28.39 ng/mL, and high specificity. Our results demonstrated the mAb had good immunological characteristics, and could provide a good antibody basis for the sensitivity and specificity immunological detection of KTI.