In this work, we demonstrate multilayer aqueous systems as media to separate gold nanoparticles of different shapes and sizes. The multilayer aqueous systems are composed of CTAB/EG solutions with different viscosities. By centrifugation, gold nanoparticles with different shapes and sizes can be dispersed in different layers of the aqueous systems. Gold nanorods synthesized by different conditions, such as changing the amount of AgNO3 or different aging times, are used to examine the multilayer aqueous systems. Gold nanorods with higher aspect ratios can be obtained by increasing the concentration of AgNO3, as confirmed by UV-Vis spectra and TEM studies. The separated gold nanorods are also characterized by surface-enhanced Raman scattering (SERS), showing lower activity for gold nanorods with higher aspect ratios, due to the size-dependent absorption of the longitudinal plasmon band. The multilayer aqueous systems can effectively separate gold nanorods prepared by different conditions and can be applied to separate other types of nanoparticles.