The immobilization of antigenic peptides in nanostructured films is promising for the development of highly specific immunosensors. In this work, we analyze the peptide p17-1 (LSGGELDRWEKIRLRPGG), derived from the HIV-1 p17 protein, immobilized into Layer-by-Layer (LbL) films and incorporated into Langmuir monolayers of phospholipids. The LbL film was assembled using different polyelectrolytes but only poly(allylamine) hydrochloride (PAH) was efficient for the peptide immobilization. The intensity in the UV-Vis. spectra of PAH/p17-1 films increased exponentially with the number of layers, which may indicate that the peptide can be reorganized in each bilayer adsorbed. Fluorescence and circular dichroism (CD) spectra indicated that the interaction with the film did not induce an alpha helix conformation in p17-1, analogously to what occurs in an aqueous solution and in contrast to the organized peptide in a methanol solution. The maximum emission for p17-1 fluorescence occurred at 340 nm in methanol, compatible with tryptophan residue buried in the solvent, while for p17-1 in an LbL film the maximum appeared at 355 nm. This red shift is consistent with the tryptophan being exposed to the environment. The CD spectra confirmed these results showing the random structure for p17-1 in the LbL films and an α-helix structure in methanol solution. The lack of structure is the probable reason for the low sensitivity toward anti-p17 observed in amperometric sensors made with PAH/p17-1 LbL films. With regard to the Langmuir monolayers, p17-1 was found to affect the surface pressure isotherms of dipalmitoyl phosphatidyl glycerol (DPPG), even at a concentration as low as 0.5 mol%. This cooperative interaction of p17-1 and DPPG may perhaps be exploited in designing new architectures for producing immunosensors based on antigenic peptides.