DNA films are of interest for use in a number of areas, including sensing, diagnostics, and as drug/gene delivery carriers. The specific base pairing of DNA materials can be used to manipulate their architecture and degradability. The programmable nature of these materials leads to complex and unexpected structures that can be formed from solution assembly. Herein, we investigate the structure of DNA multilayer films using Förster resonance energy transfer (FRET). The DNA films are assembled on silica particles by depositing alternating layers of homopolymeric diblocks (polyA15G15 and polyT15C15) with fluorophore (polyA15G 15-TAMRA) and quencher (polyT15C15-BHQ 2) layers incorporated at predesigned locations throughout the films. Our results show that DNA films are dynamic structures that undergo rearrangement. This occurs when the multilayer films are perturbed during new layer formation through hybridization but can also take place spontaneously when left over time. These films are anticipated to be useful in drug delivery applications and sensing applications.