This paper proposes a new application for natural polysaccharides (agar and carrageenan), both extracted from the cell wall of red seaweeds. Thin films were prepared by the Layer-by-Layer (LbL) self-assembly technique onto ITO (tin-doped indium oxide), where the polysaccharides of interest were deposited in layers alternating with polyaniline (PANI). The films developed were characterized by cyclic voltammetry (CV), ultraviolet-visible spectroscopy (UV-vis) and atomic force microscopy (AFM). Results showed the presence of agar as well as carrageenan, which improves the electrochemical stability of the conducting polymer in an acid medium. The interactions at the molecular level between PANI and the biopolymers affected the most appropriate sequence of deposition as employed in the process of material immobilization and also influenced the resulting morphology. Among the films studied, the most promising system as regards electrochemical measurements was the ITO/agar/PANI system, which was subsequently employed in the electrochemical detection of chromium (VI).