Abstract: The soybean FAD2-1b gene was amplified by RT-PCR from transgenic soybeans, and soybean FAD2-1b gene silencing vector was constructed for breeding transgenic soybeans with biosafety. Seed specific promoter GY1 of soybean was amplified from high protein soybean ‘Heinong 35 ’; FAD2-1b gene introns 1 was amplified from high oil soybean ‘Heinong 37’; FAD2-1b gene fragment was amplified from five transgenic acceptors soybean, it was used as forward arm and reverse arm. These target fragments were ligated to construct a double T-DNA with bar gene selection marker, promoter GY1 and FAD2-1b gene intron 1. Then double T-DNA was introduced into E. coli DH5α. The cloning of recombination plasmids was identified correctly by enzymes digestion and sequence analysis. The recombination plasmid was named pDT-FAD2I. FAD2-1b gene silencing vector has been constructed. This will constitute an important foundation for further development of FAD2-1b gene silencing soybeans and high quality soybean transgenic security.