Abstract: Duplex real-time fluorescent polymerase chain reaction technique(polymerase chain reaction,PCR)with simultaneous detection two target genes was established to screening genetically modified soybeans and deeply processed products. Multiplex primers and fluorescent probes based on the commonly used exogenous sequence of cauliflower mosaic virus 35S promoter(P-35S),Agrobacterium tumefaciens nopaline synthase terminator(T-NOS)was designed,reaction conditions and reaction systems was optimized,specificity of the duplex systems,the repeatability,sensitivity and applicability was validated to ensure two target genes could be detected with this method at the same time,with out the interference of fluorescent signals and false negative and positive results. The results indicated this method was repeatable,special and sensitive. The efficiency of amplification was between 90% and 110%,and the correlation coefficient of the standard curve R² was greater than 0.98. The determined limits for the multiplex qPCR assays was 2 copies/μL in soybean samples. This study could be widely used screening detection of genetically modified soybeans and deeply processed products with the litter reagent cost and the shorter testing time in import and export. In turn,it would provide technical guarantee for the promotion of agricultural products and food exports.