We describe a new supramolecular architecture obtained by deposition of double‐stranded deoxyribonucleic acid (dsDNA) and chitosan derivatives on a thiolated gold surface. Surface plasmon resonance was used to monitor, in real time, the construction of the supramolecular architecture. Three chitosan derivatives were used, a quaternized N‐substituted by three methyl groups in a 40.0 mol%, and two others N‐substituted with an octyl chain in a 5.0 and 25.0 mol%. The multilayer formation depends on the degree of substitution of chitosan as well as on the nature of the substituents. The adsorption of dsDNA is slower than that of chitosan. The best immobilization of dsDNA is obtained by using a quaternized chitosan and dsDNA prepared in 0.050 M acetate buffer pH 5.00. The system is stable in buffer solution independently of the nature of the chitosan derivative. The effect of the nature of the electrolyte and ionic strength is also discussed.