Iodine-131 (131I) is an essential and widely used radioisotope in thyroid diseases and animal experiments. Planar imaging has been considered the most popular method for 131I thyroid uptake radioactive activity quantification. The ROI defining section is essential and can affect the accuracy of quantitative results. However, a consistent method has not been proposed. In this study, a UC-ROI defining method based on ULWL setting and colour display grade was applied. Three steps were performed: image acquisition of five standard activity models and obtaining the exact value that the counts per radioactive activity contributes to the ROI; image acquisition of 20 rat thyroids and obtaining the counts of the ROI (thyroid); and calculating the rat thyroid radioactive activity and comparing these values with the true values. The accuracy of quantification activity of 131I in rat thyroid reached 2.62% ± 0.41%. The mean quantification within 5% could be achieved in 19 of 20 rat thyroids. No significant difference existed between calculated thyroid 131I activity and true values with a paired matched-test (t = -0.384, P = 0.706 > 0.05). The results indicated that with the UC-ROI defining method, more accurate thyroid uptake 131I radioactive activity quantification by SPECT planar imaging can be achieved in vivo rat study.