Hepatocellular carcinoma (HCC) is a common malignant cancer worldwide. Several molecules have been used as tumor biomarkers for HCC diagnosis in clinic. Besides AFP, glypican-3 (GPC3), also called DGSX, GTR2-2, MXR7, OCI-5, SDYS, SGB, or SGBS, is the one that is highly expressed in more than 70% of HCC patients.1 Although lowly expressed in melanoma, ovarian clear cell carcinoma, yolk sac tumors, neuroblastoma, and hepatoblastoma, it is silenced in breast cancer, mesothelioma, epithelial ovarian cancer, lung adenocarcinoma, cholangiocarcinoma (CCA), and normal liver tissues.2 Therefore, GPC3 might be an ideal tumor biomarker for in vivo HCC diagnosis, especially for HCC-targeted imaging. The level of GPC3 in serum or tumor tissues is usually detected by antibody-based ELISA and previously by immunohistochemical staining. However, some obvious defects of antibody have limited its wide use in vivo due to its high immunogenicity, easy degradation, and high cellular cytotoxicity effect. Therefore, a novel reagent needs to be developed as a surrogate in clinical practice. Aptamer3,4 is a kind of agent, which can specifically target varieties of polypeptides,5 proteins,6 and living cells,7 with high affinity and selectivity by virtue of its topological secondary or tertiary structure. More importantly, aptamers exhibit numerous advantages, such as easy synthetization and chemical modification, high stability, non-toxicity, and non-immunogenicity.8–11 Thus, aptamers are expected to have great in vivo application in cancer diagnosis. By far, to our knowledge, several aptamers against HCC cells have been reported, such as AS1411, a specific nucleolin-bound aptamer,12,13 and a series of 6-nt aptamers, which targeted GPC3 and were isolated by cell-systematic evolution of ligands by exponential enrichment (SELEX) containing four standard nucleobases and two added nucleobases (2-amino-8H-imidazo[1,2-a]triazin-4-one and 6-amino-5-nitropyridin-2-one, trivially Z and P), with dissociation constants (KD) in the range of 6–500 nM.14 The former was able to suppress HCC by upregulating Galectin-14, and the latter could distinguish cells that expressed GPC3 protein from those that didnot. Here, we first screened GPC3-bound single-stranded DNA (ssDNA) aptamers based on capillary electrophoresis (CE)-SELEX, then we truncated and modified candidates with locked nucleic acid (LNA) substitution and phosphorothioate linkage, and we evaluated their binding affinities on GPC3-positive and GPC3-negtive cells. AP613-1 was found specifically targeting GPC3-positive cells with a high affinity. After LNA substitution and phosphorothioate linkage, the aptamer, especially APS613-1, showed significantly increased affinity with a KD of 15.48 nM on GPC3-positive cells. In vivo imaging showed that tumor-specific fluorescent signals were clearly observed at nude mice upon HCC xenograft using Alexa Fluor 750-conjugated APS613-1 at 150 min after tail veil injection. Taken together, these results suggested that APS613-1 could be used as a probe for GPC3-positive HCC imaging in vivo.