The thyroid-stimulating hormone receptor (TSHR) plays a pivotal role in regulating thyroid growth, function, synthesis, and secretion of thyroid hormones, with its overexpression linked to various diseases, especially in tumors. A novel TSHR-targeting small molecule agonist ML-109 with a nM potency has been developed. In this study, we radiolabeled ML-109 with radionuclide iodine-131 on the quinazolin-4-one ring to prepare 131I-ML-109, and its biological performance was further evaluated as a single-photon emission computed tomography radiotracer. The results displayed that 131I-ML-109 was prepared in high radiochemical purity >95% and moderate radiolabeling yield (∼40%). Further, 131I-ML-109 demonstrated a good tumor uptake capability in both K1 and 8305C xenograft models. Overall, this study has established the synthetic strategy for preparation of 131I-ML-109, highlighting that ML-109 is a promising molecular scaffold for developing TSHR targeted imaging probe. Further structural optimization is desired to improve the diagnostic performance of the next-generation probe.