||Bio-functionalized gold nanoparticles (Au-nps) are among the most attractive nanomaterials and are widely used in nanotechnology and biomedical science. They were intensively studied as versatile reporters and carriers in developing molecular diagnostic methods, regulating intracellular gene expression, as well as building blocks for various nanostructures. One of the fundamental necessary abilities for implementing the above applications is to efficiently conjugate biomolecules to Au-nps without sacrificing either the stability of the nanoparticles or the functionality of the biomolecules. In this thesis work, we developed a novel nucleotide-mediated strategy for bio-functionalization of Au-nps that combines rapid Au-nps stabilization with facile biomolecular conjugation. Mononucleotides were found to form an adsorption layer on the Au-nps surface in a few minutes, thus stabilizing the Au-nps against salt-induced aggregation. This layer is thermally detachable, enabling its fast replacement by thiolated biomolecules. This newly developed bio-functionalization method therefore enables functionalization of the Au-nps in salt environment, unlike previous methods, with an overall synthesis time shortened significantly from hours and days to minutes without compromising the biomolecular loading on the Au-nps. Further surface density control was achieved by tuning the conjugation speed through salt concentration and by defining the conjugation time through stopping reagent introduced in a synergistic fashion. As a more advanced step towards applications, peptides were also conjugated to the Au-nps using a similar approach. Various strategies for DNA and peptide multi-functionalization on Au-nps were further investigated. The versatile function of DNA and peptide co-conjugated Au-nps was briefly demonstrated by multiplex detection of nuclease and protease using nanoparticle-based gel electrophoresis. The developed nucleotide-mediated functionalization strategy offers an attractive alternative for the preparation of bio-functionalized Au-nps. The ability to modify Au-nps with various DNA and peptide strands in a rapid, controlled and versatile manner opens up new exciting opportunities for nano- and bio- applications.