We present the formation of an ordered three-dimensional lattice of Ni nanocrystals in an amorphous alumina matrix by a self-assembly process. The self-assembled growth achieved by single-step magnetron sputtering deposition of a Ni/Al2O3 multilayer is driven by surface morphology effects. The influence of the Ni layer thickness on the Ni particle size, separation, and quality of ordering is examined. The quality of Ni nanoparticles ordering in alumina is compared to their ordering in a silica matrix. The obtained results are important for the understanding of the self-assembly process of metallic particles in amorphous matrices and the applications of such materials.