We investigated the fabrication of a 3D colloid crystal assembled in a layer-by-layer fashion on a template substrate. This method of assembling an ordered structure offers flexibility in the choice of crystal orientation, because of the template, and the capability to insert a “defect” layer to introduce defect modes in the stop bands of the crystal. The assembly of layers of 4.4 νm polystyrene microspheres on the patterned (100) substrates was studied using a combination of evaporation and chemical binding. It was observed that the ordering process depends on the physical parameters of the template such as well size and depth. A good adhesion of the beads to the template required chemical functionalization of the substrate.