Glypican-3 (GPC3) is a diagnostic biomarker for hepatocellular carcinoma (HCC). Although numerous designs targeting GPC3 have been reported, the HCC diagnostic agents with specific tumor accumulation and low background, particularly in normal liver tissue, are still in need. Peptides have attracted considerable attention as an imaging probe due to their low immunogenicity, shortin vivocirculation time, and acceptable production cost. Herein, a two-step phage display screening approach was performed against GPC3-high expression tumor xenograftsin vivo, followed by human recombinant GPC3 proteinin vitro. A GPC3-specific binding peptide, named TJ12P2, with the sequence of Ser-Asn-Asp-Arg-Pro-Pro-Asn-Ile-Leu-Gln-Lys-Arg (SNDRPPNILQKR) was identified. The apparentK(d)value between TJ12P2 and the GPC3 protein was measured as 158.2 +/- 26.25 nM. After(18)F labeling,F-18-AlF-NOTA-TJ12P2 was found accumulated in the tumors by positron emission tomography (PET) imaging in two HCC subcutaneous tumor models (HepG2 and SMMC-7721) with high GPC3 expression. Static PET imaging revealed that(18)F-AlF-NOTA-TJ12P2 accumulation in the HepG2 and SMMC-7721 tumors reached 1.825 +/- 0.296 %ID g(-1)and 1.575 +/- 0.520 %ID g(-1), with tumor-to-muscle ratios of 4.14 +/- 0.50 and 4.25 +/- 0.25, respectively, at 30 min post-injection (p.i.). Much less accumulation (0.533 +/- 0.078 %ID g(-1)) of the(18)F-AlF-NOTA-TJ12P2 was found in the control PC3 tumors with low GPC3 expression. More importantly, no obvious normal liver uptake of TJ12P2 was observed in the abovementioned animal models. As a result, a novel peptide targeting GPC3, TJ12P2, with strong affinity and specificity was identified using a two-step phage display screening technique in the present study. The(18)F-AlF-NOTA-TJ12P2 may be a promising PET imaging probe with translational potential for accurate HCC diagnosis.