The increasing data show that the soluble form of Flt-1 have the ability to block the biological activity of VEGF due to its high affinity to VEGF and formation of homologous and heterogenous dimmer with transmembrane Flt-1 and KDR. The binding activity of Flt-1 focuses on its Ⅱand Ⅲ extracellular domain. cDNA fragments encoding for signal peptide and flt-1 Ⅰ～Ⅳ extracellular domain were amplified through RT-PCR with a pair of specific primers from human umbilical vein endothelial cells. Then, the sflt-1 gene was subcloned into pcDNA3-EF-1 vector, and the expression unit of EF-1-flt-1 was subcloned into shuttle vector of pAdTrack-CMV, linearized pAdTrack-EF-1-flt-1 was co-transformed into BJ5183 cells with adenoviral genomic plasmid of pAdEasy-1. The identified recombinant DNA was transfected into 293 cells to package adenovirus. From the supernatant and cell lysis, the presence of recombinant adenovirus was proved by PCR, and RT-PCR detection demonstrated the transcription of flt-1 in MGC803 cells infected with Ad-Track-EF-1-flt-1, and immunoprecipitation reveals that soluble Flt-1 can be secreted into the culture supernatant of infected MGC803 cells. These results indicated that tumor cells infected with the prepared recombinant adenovirus vector Ad-Track-EF-1-flt-1 can secrete soluble Flt-1, which will be further used for in vivo antiangiogenesis experiments.