We investigate the temperature dependence of interactions of β-cyclodextrin (CD)/hexadecyltrimethylammonium bromide (CTAB) self-assemblies with DNA during the decompaction of DNA/CTAB complexes. By combining direct imaging techniques with density and sound-velocity measurements, we can explain the decompaction process and suggest a suitable model. The DNA-decompaction process by using CDs is accompanied by interactions with surfaces, such as glass or mica. The mechanism of β-CD/CTAB self-assembly is elucidated and the immobilization of DNA onto negatively charged surfaces is explained. Differences between the fractal dimensions of DNA that is adsorbed onto the surfaces are related to strong and weak binding, which permit the partial relaxation of DNA on the surfaces. The β-CD/CTAB self-assembled monolayers are demonstrated to be a facile and efficient route for surface functionalization, which allows for the immobilization of biomacromolecules in close proximity without any intermediate binding or deprotection steps. Moreover, this route is expected to show several advantages that might contribute to improving the performance of future biosensors as gentle immobilization-limiting alteration of the protein structure, oriented immobilization, thereby allowing homogeneous accessibility, reversible immobilization, thereby allowing reutilizations, and high compatibility with various types of biomacromolecules.