Triple-negative breast cancer (TNBC) lacks effective therapeutic targets, leading to poor prognosis. Annexin A2 (ANXA2) is overexpressed in TNBC and associated with tumor progression. This study aimed to develop a theranostic pair of 68Ga-/177Lu-labeled DOTA-conjugated octapeptide (DOTA-ANXA2-8mer2) for positron emission tomography (PET) imaging and targeted radionuclide therapy (TRT) of ANXA2-positive TNBC.Two ANXA2-targeted radiotracers-DOTA-ANXA2-8mer1 and DOTA-ANXA2-8mer2 were synthesized and labeled with 68Ga and 177Lu successfully. A series of cell assays was performed to identify the binding affinity and ANXA2 specificity in vitro. PET imaging, SPECT imaging, and biodistribution studies were performed to evaluate the pharmacokinetics in breast cancer animal models. The cancer treatment efficacy of 177Lu-DOTA-ANXA2-8mer2 was evaluated in 4T1 tumor-bearing mice.The dimer exhibited high radiochemical purity, stability, and strong affinity for ANXA2. In vitro cell uptake studies showed that the radiotracers effectively targeted ANXA2 in 4T1 and MDA-MB-231 cells, and 68Ga-DOTA-ANXA2-8mer2 showed the highest targeting efficacy. In the MDA-MB-231 tumor model, PET/CT imaging showed that 68Ga-DOTA-ANXA2-8mer2 was taken up at higher rates than 68Ga-DOTA-ANXA2-8mer1, and it exhibited favorable pharmacokinetics and safety profiles. 68Ga-DOTA-ANXA2-8mer2 was thus selected for subsequent clinical trials. PET imaging showed specific tumor uptake of 68Ga-DOTA-ANXA2-8mer2 in ANXA2-positive xenografts (4.44 ± 0.21 %ID/g at 1 h), significantly reduced by blocking. Treatment with 177Lu-DOTA-ANXA2-8mer2 (11.1 MBq) suppressed tumor growth (397.77 ± 63.72 mm3 vs. 1644.92 ± 192.56 mm3 in controls, p < 0.001) and prolonged survival (median survival: 41.2 vs 26.8 days in controls; p < 0.001). SPECT and biodistribution confirmed tumor-selective accumulation. No significant hematological or organ toxicity was observed. Immunohistochemistry revealed reduced ANXA2, CD31, and Ki-67 expression post-therapy.Two ANXA2-targeted radiotracers have been developed and evaluated in vitro and in vivo. The ANXA2 dimer exhibits satisfactory ANXA2-binding affinity and specificity, along with favorable in vivo pharmacokinetics. The 68Ga-/177Lu-DOTA-ANXA2-8mer2 theranostic pair enables non-invasive ANXA2-targeted PET imaging and effective TRT in TNBC models with minimal toxicity, supporting its clinical potential for precision management of ANXA2-positive breast cancer.Copyright © 2025. Published by Elsevier Inc.