Prostate cancer, one of the leading causes of disease and death in men all over the world, is challenging to treat. α-Enolase, a multifunctional protein, is overexpressed on human prostate carcinoma cells, and thereby it is a potential target for treatment of prostate cancer. In the current study, the pHCT74 peptide was used to construct a kind of highly targeted liposome (pHCT74-lipo) loaded with doxorubicin (pHCT74-lipo-Dox), which specifically targeted α-enolase on prostate tumour cells. Compared with liposomes without pHCT74 modification, pHCT74-lipo-Dox displayed a superior intracellular internalization with enhanced tumour cytotoxicity. In the in vivo study, pHCT74-lipo showed much higher tumour accumulation. In addition, loaded into pHCT74-lipo, doxorubicin demonstrated significantly improved anti-tumour activity on prostate tumour-bearing mice. These results suggest that the pHCT74 peptide has potential to be used in the development of a novel drug delivery system for targeted therapy against prostate cancer.