To sustain transgene expression, chitosan was studied to immobilize DNA using layer-by-layer assembly to form polyelectrolyte multilayers (PEMs). Higher DNA concentrations and longer deposition periods demonstrated more DNA adsorptions to PEMs. By adjusting pH and the molecular weight of chitosan, PEM structures were manipulated. Chitosan molecules adsorption to PEMs increased when they were at pH 6 because of their low protonation. Furthermore, the configuration of chitosan favored a coiled-form when the pH was high, as the intramolecular repulsion decreased. Therefore, interdiffusion of polyelectrolytes in PEMs was promoted to increase DNA adsorption, especially for chitosan with high molecular weight. For the release experiments, because PEMs fabricated by lower pH chitosan owned less chitosan molecules, DNA release was enhanced. However, this phenomenon did not happen to chitosan with high molecular weight, which should be due to the entanglement between polymer chains. This comprehensive approach should be beneficial to substrate-mediated gene delivery applications.