For tissue engineering concerns, gene delivery can introduce therapeutic genes to promote tissue regeneration. However, how to spatially control transfection and elongate the duration of transgene expression is a critical issue. Therefore, in this study, we applied chitosan to adsorb DNA on substrate surfaces for in situ gene delivery because chitosan is a biocompatible polycations which can be used to immobilise DNA using layer-by-layer (LbL) assembly to prepared polyelectrolyte multilayers (PEMs). DNA immobilisation can be simply controlled by bilayer numbers of PEMs so that more bilayers can deliver more DNA, which promotes not only gene delivery efficiency but also the duration of transgene expression. Chitosan solutions with different pH values were investigated for their effects on PEM constructions. The infrared spectroscopy, UV-Visible spectrometry, and quartz crystal microbalance assay demonstrated that acidic chitosan solutions reduced the ratios of chitosan adsorption but did not have significant effects on DNA deposition. The DNA release experiments suggested that PEMs with lower chitosan ratios exhibited more DNA delivery in neutral environment, however, this trend was inconsistent to the transfections results that PEMs prepared using chitosan solutions with higher pH demonstrated better transfection behaviours. To explain this contradiction, free chitosan molecules were spiked before cell seeding, by which the transfection efficiencies of the PEMs prepared by acidic chitosan solutions were highly improved, suggesting that in situ gene delivery from PEM depends not only on DNA but also on polycation release.