Abstract: Objective?To establish a strain of eukaryotic cells with overexpressed human thyrotropin receptor (hTSHR) for the development of anti-thyroid drugs. Methods?GV266 vector and hTSHR gene were digested by AgeⅠ/NheⅠ. Plasmids expressing GV266-hTSHR were constructed using T4 ligase and then transfected into 293T cells. The expression of hTSHR was determined by Western blot. Packaging plasmids were built in the 293T cells with Opti-MEM, Lipofectamine 2000 and helper plasmid. The titer of the packaging plasmids was determined with qPCR. The packaging plasmids and the plasmids expressing GV266-hTSHR were co-transfected into 293T cells to obtain a strain of cells (GV266-hTSHR-293T) with stable expression of hTSHR. The GV266-hTSHR-293T stain was detected by green fluorescent protein (GFP) fluorescence. Results? The DNA sequence of GV266-hTSHR matched that of hTSHR. The Western blot showed a 62×10 3 target band. The titer of packaging plasmids reached 2×108 TU/mL. The GV266-hTSHR-293T cells were visible under GFP fluorescence. Conclusion? HEK 293T cells with stable expression of hTSHR was established.