Thin films of alternating DNA and poly-L-lysine (PLL) layers were fabricated onto various planar surfaces by the layer-by-layer (LbL) self-assembly technique. For both the polydeoxynucleotide (PDN, polyguanylic acid or poly[G]) and the oligodeoxynucleotide (ODN, a 30-mer) investigated, UV-visible spectrometry and contact angle measurements showed that a uniform layer of DNA can be fully adsorbed onto each alternate PLL layer. Various parameters affecting the DNA loading into the composite film were investigated with a particular emphasis placed on the effect of the ionic strength in the DNA solution used for the film preparation. For the PLL/poly[G] film, it was found that 150 mug/mL of poly[G] solution containing 0.5 M NaCl attains the maximum loading for every poly[G] layer. Atomic force microscopy was utilized to measure the DNA surface density and structure at the topmost layers of several representative composite films. While the DNA film thickness increases with the ionic strength of the DNA solution, the shape of the DNA molecules adsorbed through the LbL assembly was found, for the first time, to undergo a transition from extended linear structure to a more circular or coiled configuration. The AFM images, together with results accumulated from the UV-visible spectrometric and quartz crystal microbalance experiments, helped to unravel the relationship between the DNA surface structure/loading and the ionic strength of the DNA solution. Our study clarified a possible misconception about the proportionality between DNA thickness and DNA incorporation/loading. It also provided a fundamental understanding about and the practical guidance for the utilization of the LbL method to construct DNA multilayers or polymer-encapsulated DNA molecules for gene-delivery applications.