||Gold nanoparticles (Au-nps) are one of the most attractive nanomaterials used in nanotechnology and biomedical science. The wide applications of nanoparticles are highly dependent on their size-related physical and chemical properties. In this study, we present a novel size fractionation approach for Au-nps, which combines the nucleotides-mediated stabilization mechanism for Au-nps and the size-dependent colloidal stability of nanoparticles. With nucleotides coating on Au-nps, the salt-induced aggregation of Au-nps in aqueous solutions is reversible, and the critical salt concentration (CSC) for the transition of Au-nps aggregation depends upon the size of nanoparticles: the smaller the particle, the higher is the CSC. Successful fractionations of Au-nps of 10nm, 20nm and 40nm were demonstrated in both binary and ternary mixtures with the final purity of each fraction larger than 90%. Taking advantage of the nucleotides-mediated rapid stabilization of Au-nps, the whole process can be completed within 1 hour. One more important advantage of Au-nps purified through the nucleotides-mediated approach is that they are readily conjugated with other biomolecule for further applications. In this thesis, the purified Au-nps of different sizes were modified with different substrates, e.g. DNA or peptide, for enzymatic assay. A quantitative enzyme detection approach was designed and demonstrated. DNase concentration around 0.09U/μl and trypsin concentration around 0.004mg/ml can be directly identified with naked eyes through our Au-nps based gel electrophoresis approach. Quantitative detection for multiplex enzymatic assay was also been successfully proved.