Objective To clone ORF of DcR3 gene and insert it into eukaryotic expression vector and express it in COS-7 cells. Methods Encoding sequence of human DcR3 gene was cloned by PCR and sequenced. The sequenced ORF was cloned into eukaryotic expression vector pAAV-IRES-hrGFP to construct recombinant plasmid. COS-7 cells were transfected with recombinant plasmid by lipofectamine2000. Expression of recombinant DcR3 gene was verified by Western blotting and confocal microscopy. Results A 1 000-bp gene segment was obtained by PCR and inserted into pAAV-IRES-hrGFP to construct recombinant plasmid. The gene segment was proved to be encoding sequence of human DcR3 gene by sequencing. DcR3 expression in COS-7 cells was verified by Western blotting and confocal microscopy. Conclusion DcR3 gene was successfully cloned and expressed in COS-7 cells.