Short peptide surfactants have recently emerged as a new class of amphiphiles, with tremendous potential in improving surface biocompatibility and mediating interfacial DNA immobilization. To establish their basic interfacial adsorption properties, cationic peptide surfactants V(m)K(n) have been studied by combining the measurements of spectroscopic ellipsometry (SE), neutron reflection (NR) and atomic force microscopy (AFM). Our results showed that changes in peptide structure, concentration, solution pH and ionic strength all affected their interfacial assembly. Increases in m and decreases in n reduced the critical aggregation concentration (CAC), but increased the amount of adsorption, showing the strong influence of the amphiphilic balance between hydrophilic and hydrophobic moieties. While the surface adsorbed amount increased with time and peptide concentration, an increase in ionic strength decreased peptide adsorption due to surface charge neutralization. Changes in solution pH did not affect the equilibrium surface adsorbed amount on the weakly negative SiO(2) surface, but did alter the adsorption dynamics. Neutron reflection revealed that V(6)K readily formed a bilayer structure of 35 A thickness at the interface, with the main part of the V(6) fragments being packed back-to-back to form a 15 A hydrophobic core and the two outer K regions being incorporated with a minor amount of V fragments forming the headgroup layers of 9 A each. AFM imaging revealed a sheet-like membrane structure incorporating defects of holes but the thicknesses probed by AFM were consistent with neutron reflection. It was demonstrated that the V(6)K peptide bilayer was effective for immobilization of DNA. The amount of DNA immobilized followed approximate 1:1 charge neutralization between the outer leaf peptide sublayer and the negatively charged DNA.