The fabrication of selectively functionalized micropatterns based on nanostructured porous silicon (nanoPS) by phase mask ultraviolet laser interference is presented here. This single-step process constitutes a flexible method for the fabrication of surface patterns with tailored properties. These surface patterns consist of alternate regions of almost untransformed nanoPS and areas where nanoPS is transformed into Si nanoparticles (Si NPs) as a result of the laser irradiation process. The size of the transformed areas as well as the diameter of the Si NPs can be straightforwardly tailored by controlling the main fabrications parameters including the porosity of the nanoPS layers, the laser interference period areas, and laser fluence. The surface patterns have been found to be appropriate candidates for the development of selectively-functionalized surfaces for biological applications mainly due to the biocompatibility of the untransformed nanoPS regions.