Hepatocellular carcinoma is one of the deadliest cancers in the world. In this study, a hepatocarcinoma-specific binding peptide, which could be used for drug delivery in targeting therapy, was obtained by in vivo phage display technology. After three rounds of panning, only the potential motif Pro-Ser was found in 80 sequenced phage clones. Phage A54 (sequence AGKGTPSLETTP) was shown to be the most effective and specific to the liver cancer cells by cell-based ELISA in all 130 tested clones. After phage A54 was injected i.v. into the xenograft-bearing mice for in vivo distribution, phage enrichment was found in tumor tissues compared with control phage C10 and normal liver tissues through phage titering and immunohistochemical staining. Next, the specific binding ability of synthesized peptide A54 was further confirmed by fluorescence microscopy, competition binding, and fluorescence-activated cell sorting assay. A54 and A54M (sequence AGKGTAALETTP) were synthesized and coupled to doxorubicin (DOX) to do the preliminary targeting therapy. After the treatment, the proliferation of liver cancer cells treated with A54-DOX was restrained significantly in vitro when compared with A54M-DOX-treated group. Reduction in tumor size and prolongation of long-term survival were also found in xenograft-bearing models compared with free DOX-treated group. In conclusion, the specific binding peptide A54, which was screened from phage display library, represents a promising approach for the development of novel target therapy strategies against hepatocellular carcinoma.