To screen NF-κB antagonistic drugs and research singal transduction pathway related to NF-κB, two vectors, p4κB-d2EGFP containing destabilized enhanced green fluorescent protein(d2EGFP) reporter gene and p4κB-EGFP with EGFP gene, were constructed on the base of 4 copies of NF-κB cis-element κB as enhancer, SV40 as basic promoter and neo^r gene as selective gene. The time and dose effects of d2EGFP and EGFP induced by p65 protein showed that p4κB-d2EGFP was the better NF-κB-responsive GFP reporter gene system because it is more sensitive to detect the changes of gene transcription regulation after p65 vector transiently contransfected with the two vectors respectively. The NF-κB-responsive d2EGFP clonal cell line named HEK-d2EGFP was established after p4κB-d2EGFP stablely transfected into HEK 293 cells. With the cotransfection of NF-κB transcription factor decoy (TFD) and p65 vector into HEK-d2EGFP cells, the results showed the groups of 1 mg/L and 2 mg/L TFD could antagonized the d2EGFP expression induced by p65 protein significantly. It was demonstrated that the NF-κB-responsive d2EGFP reporter system could report and detect NF-κB activation accurately and dynamically.