Quantitative determination of the density and conformation of DNA molecules tethered to the surface can help optimize and understand DNA nanosensors and nanodevices, which use conformational or motional changes of surface-immobilized DNA for detection or actuation. We present an interferometric sensing platform that combines (i) dual-color fluorescence spectroscopy for precise axial co-localization of two fluorophores attached at different nucleotides of surface-immobilized DNA molecules and (ii) independent label-free quantification of biomolecule surface density at the same site. Using this platform, we examined the conformation of DNA molecules immobilized on a three-dimensional polymeric surface and demonstrated simultaneous detection of DNA conformational change and binding in real-time. These results demonstrate that independent quantification of both surface density and molecular nanoscale conformation constitutes a versatile approach for nanoscale solid-biochemical interface investigations and molecular binding assays.