Obtaining marker-free plants by high efficiency will benefit the environmental release of transgenic crops. To achieve this point, a binary vector pNB35SVIP1 with three T-DNAs was constructed by using several mediate plasmids, in which one copy of bar gene expression cassette and two copies of VIP1 gene expression cassette were included. EHA101 Agrobacterium strain harboring the final construct was applied to transform soybean (Glycine max) cotyledon nodes. Through 2～3 months regeneration and selection on 3～5mg/L glufosinate containing medium, transgenic soybean plants were confirmed to be obtained at 0.83%～3.16%, and co-transformation efficiency of both gene in the same individual reached up to 86.4%, based on southern blot test. By the analysis of PCR, southern blot and northern blot combining with leaf painting of herbicide in T₁ progenies, 41 plants were confirmed to be eliminated of bar gene with the frequency of 7.6%. Among the ₁ populations tested, the loss of the alien genes happened in 22.7% lines, the silence of Tbar gene took place in 27.3% lines, and VIP1 gene silence existed in 37.1% marker-free plants. The result also suggested that the plasmid with three T-DNAs might be an ideal vector to generate maker-free genetic modified organism.