HER2, a cell membrane protein overexpressed in many types of cancers, is correlated with poor diagnosis, suboptimal treatment outcome, and low survival rate. Multiple HER2-targeted drugs have been developed for the treatment of HER2-overexpressing tumor, which can in turn down-regulate HER2 expression. It is thus significant to profile HER2 expression for cancer prognosis, patient stratification, and monitoring therapy response. Aptamers, a class of single-stranded DNA/RNA (ssDNA/ssRNA) ligands, are promising for molecular biomarker imaging. Aptamers typically have strong binding affinity, high selectivity, batch-to-batch reproducibility, and low toxicity, and systemically injected aptamers often have high tumor-to-background ratio within a short time. However, current aptamers have been mostly screened in vitro, and these aptamers may lose binding ability in vivo due to conformational change under physiological environments. Here, a DNA library was combinatorially screened in vitro and in vivo, to select HER2-targeting DNA aptamers, termed Heraptamers, and labeled with 18F for positron emission tomography (PET) imaging of HER2 in ovarian cancer. Specifically, using systematic evolution of ligands by exponential enrichment (SELEX), Heraptamer candidates were first selected and validated in vitro using HER2 extracellular domain (ECD) and HER2-positive SKOV3 cancer cells; then, aptamer candidates were modified with alkyne, radiolabeled with 18F using azide-functionalized precursors by click chemistry, and screened in SKOV3-tumor-bearing mice using PET. Two aptamers, Heraptamer1 and Heraptamer2, reached high tumor uptake ratios within as short as 1 h. At 1.5 h post injection, the tumor uptake ratio of these two aptamers was up to 0.5%ID/g (injection dose/gram tissue), with tumor-to-muscle ratio of 4.55 ± 1.63 in SKOV3 tumor. In contrast, these aptamers have low uptake ratios in control MDA-MB-231 tumors. These preclinical studies showed that Heraptamers are promising for specific HER2 imaging.