<p>[¹⁸F]-Fluoroestradiol, which is recently approved by the FDA, is a well-recognized estrogen receptor radiopharmaceutical in nuclear medicine to investigate both primary and metastatic breast cancers. This study introduces an innovative and effective method for synthesizing the DOTA-coupled estradiol derivative and evaluating its preclinical potential. ⁶⁸Ga/¹⁷⁷Lu-labeled estradiol derivative was evaluated in vitro for its stability in human plasma, and binding capacity was determined using the ER-positive MCF-7 breast cancer cell line. In vivo, biodistribution and tumor targeting were conducted in ER-positive MCF-7 tumor-bearing nude mice. DOTA conjugated estradiol was prepared conveniently using solid-phase synthesis, and its radiolabeling with both ⁶⁸Ga and ¹⁷⁷Lu resulted in the formation of one major product, with high labeling efficiency (≥ 90%). Also, a high stability of the radiotracer was found in human plasma. The radiolabeled estradiol derivative exhibited a nanomolar affinity (&lt; 20 nM) specific to the MCF-7 cell line. In the MCF-7 tumor xenograft model, the radioestradiol derivative displayed fast clearance from the blood and excretion mainly by the renal system. A rapid accumulation of 3.79% IA/g was observed in the MCF-7 tumors at 1&#xa0;h p.i., whereas a low to moderate retention of radioactivity was seen in the normal organs, including the heart, lungs, liver, stomach, spleen, intestines, and kidneys (&lt; 5% ID/g). The receptor specificity of the radioestradiol was confirmed by the receptor-blocking assay. The rapid and efficient targeting of tumors, along with the favorable pharmacokinetics, emphasizes the potential of radioestradiol for targeting tumors. Additionally, PET imaging demonstrated good visualization of MCF-7 tumors in nude mice. Our results indicate that the ⁶⁸Ga/¹⁷⁷Lu-labeled estradiol derivative could serve as a promising radiopharmaceutical option for the effective targeting of MCF-7 tumors.</p>