Gold nanoparticles conjugated with DNA represent an attractive and alternative platform for broad applications in biosensors, medical diagnostic, and biological analysis. However, current methods to conjugate DNA to gold nanoparticles are time-consuming. In this study, we report a novel approach to rapidly conjugate DNA to gold nanoparticles (AuNPs) to form functional DNA/AuNPs in 2-3 h using Tween 80 as protective agent. With a fluorescence-based technique, we determine that the DNA density on the surface of AuNPs achieves about ∼60 strands per particles, which is comparable to the loading density in the current methods. Moreover, the DNA/AuNPs synthesized by our approach exhibit an excellent stability as a function of temperature, pH, and freeze-thaw cycle, and the functionality of DNA/AuNPs conjugates is also verified. The work presented here has important implications to develop the fast and reproducible synthesis of stable DNA-functionalized gold nanoparticles.