In the design of DNA-based hybrid devices, it is essential to have knowledge of the structural, electronic and optical properties of these biomolecular films. Spectroscopic ellipsometry is a powerful technique to probe and asses these properties. In this chapter, we review its application to biomolecular films of single DNA bases and molecules on silicon and diamond surfaces characterized in the spectral range from the near-infrared (NIR) through the visible (Vis) and toward the vacuum ultraviolet (VUV). The reported optical constants of various DNA structures are of great interest, particularly in the development of biosensors.