In this study, two types of BolA-like amphiphilic peptides with dual ligands comprising a tumor-targeting moiety of RGD sequence and a cell-penetrating moiety of R8 sequence are designed and synthesized as gene vectors. The BolA-structural peptide carriers can self-assemble into spherical nanoparticles with a hydrophilic core and shell, which are similar to the viral capsid and can bind plasmid DNA in an aqueous medium to form viral-mimetic complexes. It is found that the BolA-like dual ligands system exhibits significantly enhanced gene expression in both HeLa and 293T cell lines, as compared with poly(ethylenimine) PEI. These BolA-like amphiphilic peptides are promising in clinical trials of gene therapy.