In this study, cationic and amphiphilic oligopeptides, (A)12(H)5(K)10 (AK27) and (A)12(H)5(K)15 (AK32), were designed and tested as a nonviral gene vector. The oligopeptides had critical micelle concentrations (CMC) of 0.9 and 1.1 mg mL−1 respectively, indicating that they were able to self-assemble into core–shell nanoparticles at concentrations above the CMC value, which was confirmed by electron microscopy. The nanoparticles were cubic and had an effective diameter of 700–900 nm, as well as a zeta potential of 18 to 20 mV. The formation of nanoparticles increased the local concentration of cationic charge in the solution, leading to improved DNA binding ability and better protection from enzymatic degradation compared to the control peptide (H)5(K)10 (HK15) without a hydrophobic block. The smallest effective diameters of AK27–DNA and AK32–DNA complexes were 442 and 332 nm, respectively, and the highest zeta potentials were 7.8 and 18.2 mV, respectively. In comparison, HK15 formed much larger DNA complexes with a nearly neutral zeta potential. Cytotoxicity tests showed that HEK293, HepG2, and 4T1 cell lines had viabilities of more than 80% after incubation with AK27 and AK32 peptides, which was much higher than that obtained from polyethyleneimine (PEI) incubation. Furthermore, an increased length of lysine block, and the presence of the hydrophobic block did not show any significant increase in cytotoxicity. More importantly, AK27 and AK32–DNA complexes induced much higher luciferase expression efficiency than KH15 in all three cell lines tested. AK32 with a longer lysine block led to more efficient gene expression than AK27. In addition, the gene-expression levels mediated by AK32 nanoparticles were comparable to those provided by PEI, especially in HepG2 and 4T1 cell lines. These cationic nanoparticles made from biodegradable and biocompatible peptides may provide a promising carrier for gene delivery.