Thin films of alternating DNA and rare earth ion Eu3+ layers from dilute aqueous solutions were fabricated onto quartz substrates and silicon wafers through the layer-by-layer (LbL) self-assembly technique. UV-visible spectroscopy shows that a uniform layer of DNA can be fully adsorbed onto each alternate Eu3+ layer. Microscopic FTIR spectra show Eu3+ interacts with both the phosphate groups and nitrogenous bases of DNA, and the formation of {DNA/Eu}(n) films induces a change of the conformation of the DNA secondary structure to a certain extent. Various parameters affecting the DNA or Eu3+ loading into the composite film were investigated with emphasis on the effect of the pH and ionic strength of the DNA solution used for the film preparation. Atomic force microscopy was utilized to observe the morphologies of the DNA in the films obtained at two different pH values. Small molecules, such as alpha-tenoyltrifluoroacetone (TTA), Hoechst 33258 (Hoe), and ethidium bromide (EB), are all observed to interact with Eu3+ or DNA in the {DNA/Eu}(n) films. The {DNA/Eu}(n) films incorporated with these molecules show different fluorescent characteristics, and the fluorescence intensity of the films versus the bilayer number has a good linear relationship, confirming the potential for creating a different luminescence ability of the multilayer by controlling the number of DNA/Eu bilayers.