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In this work, we present a novel approach for the size fractionation of gold nanoparticles (Au-nps), which combines the nucleotide-mediated stabilization mechanism for Au-nps to the size-dependent colloidal stability of nanoparticles. Under nucleotide-coating, Au-nps undergo reversible salt-induced aggregation in aqueous solutions, while the critical salt concentration (CSC) for the transition of Au-nps aggregation increase as the nanoparticle size getting smaller. Successful fractionation of both binary and ternary mixtures composed of Au-nps in 10 nm, 20 nm and 40 nm were demonstrated and the final purity of each fraction is larger than 90%. The whole process can be accomplished within one hour due to the rapid stabilization of Au-nps with nucleotide-mediation.