A novel self-assembly technique, analogous but different in essence from the conventional gas–liquid interface self-assembly method, is proposed for the first time to fabricate three-dimensional (3D) colloidal crystals. This facile, efficient, and cost-effective technique is designated as the suspending self-assembly method (SSAM). Water is used as the solvent in the self-assembly of colloidal crystals. Purely by manipulating the electrostatic intersphere repulsive forces with deionization and concentration, a variety of polystyrene (PS) colloidal crystals with a broad range of diameters are successfully prepared. Furthermore, large-scale preparation of such 3D colloidal crystals with a high degree of order is easy to achieve by this new approach. The self-assembly mechanism of 3D colloidal crystals in a water medium is subjected to in-depth investigation, and quantitative calculations are conducted to ascertain the difference from the traditional gas–liquid interface assembly method. It is found that electrostatic repulsive forces between PS microspheres, rather than capillary forces, work as the major driving forces for the successful ordering process. This work exerts great potential for revolutionizing the current preparation techniques of colloidal crystals.