Soyabean Kunitz-type trypsin inhibitors are important antinutrient factors. The full-length DNA fragment of soyabean Kunitz-type Trypsin Inhibitor gene (KSTI3) was amplified by PCR technique, then the PCR products were cloned into pMD18-T vector. The nucleotide sequence analysis showed that the cloned fragment consisted of 654 bp and shared 99% identity with the reported KSTI. The fragment of antisense gene + plus sense gene was constructed to the downstream of 35 S promoter in the binary vector pBI121, which formed the recombinant plasmid pBIKSTI3. The plasmid pBIKSTI3 was mobilized into Agrobacterium tumefaciens strain EHA105 by freeze-melting methods, and a plant expression vector was obtained.