The introduction of surfactants to stabilize colloidal citrate-reduced gold nanoparticles (prevent aggregation) is usually used in surface-enhanced Raman scattering (SERS) applications. However, the surfactants have many drawbacks for SERS applications, such as increasing the SERS background and blocking surface active sites. Here, we develop a surfactant-free method to stabilize colloidal cit-AuNPs based on alkali regulation, and this method can prevent gold nanoparticle aggregation under different harsh treatments, including ligand modification, centrifugation-based washing/enrichment, and salt addition. The SERS spectra, density functional theory simulation, and ζ potentials of cit-AuNPs indicate that the stability of enhanced cit-AuNPs under alkaline conditions is attributed to both the increased negative charge density (by ∼6 times from pH 7 to 12) and the molecular configuration on the metal surface. Compared with surfactant-based methods, this method can well maintain the inherent optical and interface properties of nanoparticles, avoid the SERS background, and avoid blocking of the surface active site due to the presence of surfactants. This method will enable AuNPs to have a wide range of applications in areas such as highly sensitive SERS sensors.