Nuclear factor kappa B (NF-κB) plays an important role in regulating immune response and inflammation. In order to obtain the NF-κB promoter reporter gene and further study the mechanism of the immune response in primitive vertebrates, based on the CDS sequence of the NF-κB gene previously cloned from Lampetra morii, we obtained the L. morii NF-κB promoter sequence by using the 5′upstream promoter characteristic sequence of the NF-κB from L. japonica, which is highly homologous to L. morii, as a template to design primers. The cloned NF-κB promoter sequence from L. morii was 3 169 bp, which was submitted to NCBI Genbank to obtain accession number MN368861. The sequence is predicted by AliBaba2.1 software to include CREB (cgtgacttca), Oct-1 (aatatgaatt), GATA-1 (gaacctatct), AP-1 (ggttgagtca), NF-kappa B (ctttcctgtt), IRF-1 (tttcctgttc), Sp-1 (tgtgaggggt), c-Jun (acgtgacttc), c-Fos (ggttgagtca) and many other transcription factor binding sites, and contains TATA box transcription core elements. The MethPrimer software predicts that there are CpG islands with a CG content greater than 50% and a length of 196 bp at the 1 207-1 402 bp sequence. pGL3-NF-κB-pro recombinant plasmid was constructed by double digestion technology and transfected into human embryonal kidney (HEK293T) cell and epithelioma papulosum cyprinid (EPC) cell. The results of double luciferase reporter gene system assay showed that the fragment sequence had promoter activity in both mammalian cell lines and fish cell lines, while the promoter activity increased significantly after Toll-like receptors (TLR) ligands LPS stimulation in HEK293T cell. In this study, the NF-κB reporter gene of L. morii was successfully constructed, and its activity was confirmed in different cell lines. The results showed that NF-κB is involved in the immune response of TLR signaling pathway after LPS stimulation in L. morii. The construction of the NF-κB reporter gene of L. morii laid a foundation for exploring the signaling mechanism of the primitive vertebrate immune system.