The invention relates to a method for separating nano-particles at a water-phase density gradient centrifugation rate, which mainly comprises the following steps: 1) preparing the nano-particles into a homogeneous transparent colloid nano-particle solution by means of ultrasound, stirring and the like; 2) preparing density gradient solutions with different mass percentage concentrations respectively; 3) adding certain amount of density gradient medium solutions with different concentrations into a centrifuge tube in turn to prepare a step-like or linear density gradient solution; and 4) adding the solution containing colloid nano-particles to the liquid surface of the density gradient solution slowly, and performing centrifugalization under certain condition. Because the colloid nano-particles with different sizes have different sedimentation rates in the density gradient solution and can be detained at different positions in the density gradient solution so as to achieve the effect of separation. The method has the advantages of simple method, quick separation, low cost, small influence on the stability and the purity of a sample, and the separation effect of certain section can be enhanced selectively by adjusting and controlling centrifugal parameters.