Gold nanoparticles (AuNPs) have found widespread applications in life sciences. While synthesis of monodispersed AuNPs has been fairly convenient by using chemical reduction of chloroauric acid by sodium citrate, we found that AuNPs of high quality and high concentrations were not readily obtained via this method. As an example, we showed that monodispersed 13-nm AuNPs were readily synthesized at relatively low concentrations ( e. g. 3.5 nM); in contrast, 13-nm AuNPs of 17 nM obtained by the direct reduction method were irregularly shaped and not well dispersed. In this work, we demonstrated that AuNPs of high concentration could be prepared by a two-step approach, i.e. chemical reduction at low concentrations and subsequent centrifugation. Compared to the direct reduction method, this new two-step method led to AuNPs with high salt resistance and high stability, which are essential for the preparation of DNA-AuNPs conjugates for DNA biodetection.