Objective To prepare and verify a recombinant baculovirus harboring the human sodium iodide symporter (hNIS) under control of the early growth response-1 (Egr-1) promoter, and to provide the foundation for the research on radionuclides gene targeted treatment of malignant tumors. Methods The vector pFB was cut from plasmid pFB-CMV-EGFP; Egr1 was cut from plasmid PGL3-Egr1-luc; and hNIS was cut from plasmid pcDNA3.1-CMV-hNIS. Then recombinant baculovirus plasmid pFB-Egr1-hNIS was constructed. In addition, a positive control plasmid pFB-CMV-hNIS and a control plasmid pFB-0 were constructed. Baculovirus Bac-Egr1-hNIS and its controls were prepared, verified, amplified, and collected for titer determination. After glioma cell line U87 was infected with Bac-Egr1-hNIS and exposed to ¹³¹I, the presence and enhanced expression of hNIS protein was tested by immunofluorescence using anti-hNIS antibody. To verify the function of hNIS after ¹³¹I stimulation, the uptake of iodide was determined after incubation of the processed cells with ¹²⁵I. Results Recombinant plasmid pFB-Egr1-hNIS, pFB-CMV-hNIS, and pFB-0 were correctly constructed and confirmed by DNA sequencing. Transposition was correct and recombinant Bacmid was extracted. The recombinant baculovirus were packed, verified, and its titer was about 1×10¹⁰ Pfu/mL. After incubation with 3 MBq/mL ¹³¹I, immunofluorescence showed that the hNIS expression level of Bac-Egr1-hNIS infected U87 cells increased dramatically. In another side, ¹³¹I stimulated U87 cells accumulated more ¹²⁵I than did non stimulated cells. Conclusion The recombinant baculovirus Bac-Egr1-hNIS and its controls were constructed and verified, and high titer was obtained. This study provides a foundation of radiosensitive positive feedback system for further study.