The electronic, optical, magnetic, and catalytic properties of noble metal nanoparticles (NPs) strongly depend on their size. The size control of the noble metal NPs is one of the significant issues in the field of nanotechnology. We demonstrate both theoretically and experimentally that gold NPs can be separated by density gradient centrifugation (DGC) according to their size. By analyzing the force balance in a centrifuge with a horizontal rotor, a model was developed that relates the sedimentation shift to the centrifugation time and can be used to predict the effect of separation by the shift of particles within a specific period of time. The influences of centrifugation time and centrifugal force on the separation effect of DGC were evaluated.