Clay minerals are suitable matrices to anchor organic molecules such as antimicrobial peptides (AMPs) so that their bioactivity is maintained, enabling the formation of new materials with potential for new applications in biotechnology. The objective of the present study was to develop a nanostructured film where the properties of palygorskite (Plg) were combined at the molecular level with Dermaseptin 01 (DRS 01), in which the clay mineral also served as a substrate for the immobilization of this peptide. The films were prepared using the Layer-by-Layer (LbL) self-assembly technique. Crude palygorskite without purification (Plg IN) was subjected to physical and chemical procedures to increase its adsorptive properties. The structure, chemical composition, and morphology of Plg were investigated by X-ray diffraction (XRD), Fourier-transform infrared spectroscopy (FTIR), X-ray fluorescence spectrometry (XRF), scanning electron microscopy (SEM), and transmission electron microscopy (TEM). LbL films were adsorbed onto ITO (Indium Tin Oxide) and characterized electrochemically by cyclic voltammetry (CV), UV-Visible spectroscopy, and atomic force microscopy (AFM). For the ITO/DRS 01 and ITO/Plg/DRS 01 films, an oxidation process at +0.77 V was observed, confirming that the DRS 01 maintained its electroactive behavior and intrinsic properties. The results also showed that Plg served as excellent support for the immobilization of DRS 01, increasing its concentration and availability in the film form. This work reported immobilizing the DRS 01 peptide with Plg for the first time in an ultrathin film with bioactive properties. Thus, the film developed can be explored for applications such as biosensor devices and antimicrobial coating materials as well as other biotechnological applications.