Thiolate-protected gold nanoclusters with high chemical stability are exploited extensively for fundamental research and utility in chosen applications. Here for the first time, the controlled destabilization of extraordinarily stable thiolated gold clusters for the growth of single-crystalline gold nanoparticles (AuNPs) is demonstrated, which was achieved simply via the oxidation of surface-protecting thiolates into disulfides by hydrogen peroxide under basic condition. By combining with our experimental observations over the entire destabilization and growth process, the new growth mechanism from clusters to AuNPs is revealed by density functional theory (DFT) calculations. It is found that the size of AuNPs decreases with the increase of hydrogen peroxide concentration due to the generation of more nuclei at the higher hydrogen peroxide concentrations. In addition, the preparation of AuNPs is tuned by changing the concentration of hydrogen peroxide, and they are self-assembled into microspheres via an evaporation-mediated process, which can induce strong plasmonic coupling between adjacent AuNPs for ultrasensitive surface-enhanced Raman scattering detection. The present work demonstrates a facile route to functionalize and engineer AuNPs via controlling the reaction conditions and the ratio of precursors, and thus bring new possibilities for using more clusters as precursors to construct novel nano/microstructures for various applications.