<p>HER2 is a well-established target for antibody-based drug delivery. Single-domain antibodies (sdAbs) offer advantages over monoclonal antibodies, including faster clearance, higher antigen-binding affinity, and improved tumor penetration. This study investigates the biodistribution of HER2-targeting sdAbs (with and without HIS-tag) labeled with ⁶⁸Ga and their therapeutic efficacy radiolabeled with ¹⁷⁷Lu, in HER2-positive xenograft. Mice received 10 MBq of ⁶⁸Ga-labeled HIS-tagged sdAb and underwent PET/CT scans immediately and three hours post-injection. The following day, untagged radioactive sdAb was administered and scanned similarly. Controls were co-injected with an excess of unlabeled sdAb. For therapy study, mice received either four weekly injections or a single dose of ¹⁷⁷Lu-labeled sdAb (without HIS-tag). Radiotracers were synthesized with high radiochemical purity (&gt; 99.8%). PET/CT imaging revealed specific tumor accumulation and low background, except in excretory organs. Co-injection of unlabeled sdAbs reduced tumor uptake, confirming tracer specificity. ¹⁷⁷Lu-labeled sdAb therapy prolonged median survival by 34 d (single dose) and 26 d (fractionated therapy) versus respective control group treated with unlabeled sdAb. The fractionated therapy group exhibited significantly lower tumor volumes and did not exceed 200% of the initial tumor volume. ⁶⁸Ga-labeled sdAbs enabled high-contrast PET imaging and ¹⁷⁷Lu-labeled sdAbs, particularly in fractionated regimens, effectively delayed tumor progression and were well tolerated, supporting further clinical translation.</p>