ABSTRACT Bowman-Birk inhibitor (BBI) is the main type of soybean trypsin inhibitor, which is a kind of antinutritional factor. It can cause indigestion and hinder growth and development of human and animal. Herein, a rapid immunochromatographic strip was developed in a sandwich format using the anti-BBI monoclonal antibody (mAb) in order to specifically detect BBI. The mAbs showed high affinity towards BBI and no cross-reactivity with other soybean allergenic proteins and antinutritional factors. The assay could be performed within 10 min. The limit of detection of the test strip was 0.5 µg/mL for visual and 0.23 µg/mL for strip reader. The assay showed high specificity for BBI and recovery efficacy of BBI in spiked milk samples ranged between 83.24% and 89.86% with the coefficient of variation of less than 7.98%. Therefore, the test strip could be used as a rapid and reliable detection method for BBI qualitative or quantitative assessment.