Objective To construct a green fluorescent protein expression plasmid pEGFP-C3-anti-TAG72 scFv-CD28, containing anti-TAG72 single chain variable fragment (scFv) fused into the transmembrane and intracellular domain of the signal-transducing chain of CD28 gene, and to transfect it into peripheral blood mononuclear cells． Methods Recombinant transmembrane and intracellular domain of CD28 cDNA and anti-TAG72 scFv cDNA fragment was subcloned into pEGFP-C3 vector. Recombinant clones were selected by Kanamyein， and then identified by PCR， enzyme digestion analysis and DNA sequencing. The recombinant plasmid was transfected into peripheral blood mononuclear cells by means of lipofection. The recombinant protein expression was confirmed by immunocytochemistry, laser scanning confocal microscope, PCR and Western blot analysis. Results The fused gene fragment of anti-TAG72 scFv-CD28 was successfully inserted into pEGFP-C3 plasmid, and it was confirmed by enzyme digestion and DNA sequencing. The fused anti-TAG72 scFv-CD28 gene and its protein was identified in peripheral blood mononuclear cells． Conclusion The eukaryotic expression plasmid pEGFP-C3-anti-TAG72 scFv-CD28 was successfully constructed and transiently expressed in peripheral blood mononuclear cells， which would lay a foundation for further studies on the role of it to activate tumor-associated antigen-specific T lymphocyte, for generating of modified T lymphocytes targeting gastrointestinal tumors.