Nanoparticles possessing functional groups that can be readily conjugated (e.g., through click chemistry) are important precursors for the preparation of customized nanohybrid products. Such nanoparticles, if they are stable against agglomeration, are easily dispersible and have well-defined surface chemistry and size. As click-ready reagents, they can be stored until their time of use and then simply dispersed and reacted with an appropriate substrate. Gold nanoparticles (AuNPs) are excellent candidates for this purpose, and some clickable gold nanoparticles have been developed; however, AuNPs for use in aqueous systems are often prepared through difficult multistep processes and/or can be poorly dispersible in water. Here we report a single-step synthesis of clickable, water-dispersible AuNPs. The synthesis yields uniform, 3.5 nm diameter cores coated with a well-defined molecular ligand shell that makes the AuNPs stable and dispersible in water. The AuNP mixed ligand shell consists of hydroxyl-terminated ethylene glycol-based ligands to promote dispersion in water and a small number of azide-terminated ligands that readily undergo click reactions with alkynes. The use of a mesofluidic reactor affords fine control over the core size and ligand shell composition and ensures reproducible results (e.g., less than 0.1 nm variation in core diameter between batches). The purified reagents were successfully coupled to a variety of alkyne-containing substrates using both Cu-catalyzed and strain-promoted click reactions. Particle size, morphology, stability, and surface composition were thoroughly characterized using small-angle X-ray scattering, transmission electron microscopy, X-ray photoelectron spectroscopy, UV-vis, and 1H NMR before and after the click reactions. Both the parent nanoparticles and their click chemistry products are stable during storage and remained dispersible for over a year in water, suggesting their potential for environmental, biological, and biomedical applications.