Using genetic engineering technology, the green fluorescent protein (GFP) reporter gene plasmid was constructed, which is convenient for live-cell imaging, Western blot (WB), cell flow analysis and live-cell tracing, so as to realize the screening of anti-tumor drugs. The promoter sequence of NF-κB gene and GFP gene fragment were amplified by polymerase chain reaction (PCR), cloned into the pGL6-Enhancer vector, and the plasmid construction was proved by colony PCR, digestion identification and sequencing. The successful plasmid is then transfected into HEK-293T cells, and the reported plasmid is normally expressed within the cell based on live-cell fluorescence imaging, WB and flow cytometry. Finally, the anti-tumor drugs rapamycin, paclitaxel, gemcitabine, and the peptide anti-tumor drugs M1-20 and M1-21, which were being developed in our laboratory, were selected to verify that the gene system can respond to the effects of the drug on cells. The establishment of this drug screening platform provides a useful tool for studying the effects of anti-tumor drugs on cells, and also provides materials for building a live cell tracer system.