[Background] Brucellosis is a zoonotic infectious disease caused by Brucella spp. and threats the development of animal husbandry and human health. A novel plasmid was constructed for promoter activity determination in Brucella based on NanoLuc luciferase gene (nluc), which is important for research on regulatory mechanism of Brucella virulence genes. [Objective] Preparation of rabbit polyclonal antibody of Nluc, construction of a Nluc reporter plasmid for promoter activation determination in Brucella, and verification of the Nluc reporter plasmid for Brucella bcsp31 gene and virB promoter. [Methods] The nluc gene was ligated into prokaryotic expression vector pET-28a and constructed as recombinant vector pET-Nluc. The New Zealand rabbit was immunized to prepare polyclonal antibody of Nluc protein. The plasmids pNluc, pBcsp31-Luc and pVirB-Luc were constructed based on a broad-host-range vector pBBR1MCS. Brucella recombinant strains S2308(Nluc), S2308(Bcsp31-Nluc) and S2308(VirB-Nluc) were constructed by electrotransformation of plasmids. The promoter activity of bcsp31 and virB were detected in TSB. The activity of virB promoter in Brucella was compared in TSB and within RAW264.7 cells. [Results] The Nluc protein was expressed and purified. The ELISA titer of polyclonal antibody was approximately to 1:100 000. The plasmid pNluc, pBcsp31-Luc, pVirB-Luc and the S2308(Nluc), S2308(Bcsp31-Nluc), S2308(VirB-Nluc) strains were constructed successfully. The results of bcsp31 and virB promoter activity in TSB showed that promoter activity can be detected accurately in pNluc plasmid. The result of virB promoter activity in intracellular Brucella showed that the activity of virB promoter is enhanced significantly. [Conclusion] In this study, a plasmid for promoter activity determination of Brucella genes was constructed successfully. The results of this study showed that the promoter activity of Brucella genes can be detected accurately in pNluc plasmid. This study provides a novel strategy for determining promoter activity of Brucella virulence genes, which may benefit investigation of Brucella pathogenesis.