Functionalized DNA molecules have garnered significant interest due to easy implementation of specific functions into DNA via specific nanomaterials and the novel characteristics of DNA molecules. The preparation of nanomaterial-doped mull-layered DNA thin films with various nanomaterials and their resulting physical characteristics is rarely discussed. We fabricated individual (e.g., Co2+) and mixed (e.g., Cu2+ and Tb3+ mixed) ion-doped mull-layered (Le., single-, double-, triple-, quadruple-, and octuple-layered) DNA thin films and examined their layer-dependent physical characteristics. Successive spin coating was used to obtain the desired number of layers and incorporated various functionalities to each layer by embedding different dopant ions. Metal (e.g., Co(2+ )and Cu2+) and lanthanide ions (e.g., Eu3+ and Tb3+), which possess unique intrinsic characteristics, were embedded into the DNA by chemical intercalation and electrostatic interactions. To understand the physical characteristics of ion-doped mull-layered DNA thin films, X-ray photoelectron spectroscopy (XPS), Raman, absorption, and photoluminescence (PL) were used to examine the chemical interactions and energy transfer between the DNA and dopants. We observed layer-dependent PL quenching effects in the Co2+ -doped DNA thin film, which served as a PL blocking layer. Current-voltage, capacitance, and dielectric constant of each ion-doped DNA thin film were measured to determine their electric characteristics. The ion-doped DNA films exhibited either p-type or n-type semiconductor behaviour depending on the type of dopant ion and the dielectric constant of ion-doped DNA thin films differed from that of pristine DNA. The technique developed herein can be used to implement multiple functionalities into DNA thin films by simple doping with desired ions and stacking different functional layers to enhance specific functionalities with high efficiency.