The interest in DNA-protein-based diagnostics has recently been growing enormously, which makes the separation process of DNA or protein from a cell extract extremely important. Unlike the traditional separation process, a novel approach is in demand which can nondestructively isolate the target biomolecules without sacrificing the other components in the mixture. In this study, we have demonstrated a new and simple separation technique by using well-established bifunctional Au-Fe3O4 nanocomposites as the separation nanoprobes to efficiently isolate the specifically targeted nanomolar concentrated DNA over 70% from its associate DNA-protein mixture in the presence of a magnetic field. The sensing accuracy of both as-separated DNA and protein are quantitatively examined by UV-vis spectroscopy, and then qualitatively validated by gel analysis. Results obtained in this study clearly demonstrated that this newly developed separation procedure not only provides the efficient separation for the targeted DNA but can also maintain the bioactivity of as-separated protein and DNA solutions. The superiority of this technique can open an avenue to establish a label-free and nondestructive platform for a wide variety of biomolecule separation applications.