Patterning of many types of biomolecules over length scales ranging from micrometers to nanometers is of great interest for biosensors, cell culture dishes, medical implants and tissue engineering. Ideally these devices require attachment of biomolecules at specific locations on solid substrates with precisely controlled chemistry, but to function fully the non-specific adsorption in surrounding regions must be prevented. Currently, the most widely used techniques for patterning are photolithography, soft lithography and electron beam lithography, all of which involve multi-step surface modification directly onto substrates, and are time consuming and expensive. We have shown recently that highly ordered binary colloid patterns, with controllable dimensions, can be generated from simple self-assembly of large and small particles onto surfaces, where single layers of large particles are surrounded by crystals of smaller particles. In addition, when the particles are pre-coated with proteins (e.g. albumin, lysozyme and antibodies) the assembly process also takes place. This opens up the possibility of patterning many proteins on one substrate with controllable dimensions and high order. The crystals are also used to generate chemical patterns since the large particles act as a mask during, for example sputtering of Au, since the region in contact with the substrate remains uncoated. The thickness of gold features can be controlled by the sputtering time. We demonstrate that the resultant Au layer can be coated with a protein resistant mercapto-oligo(ethylene glycol) layer ((1-mercapto-11-undecyl)-tri(ethylene glycol)) that allows selective adsorption of fluorescently labelled proteins, such as FITC-labelled antibodies or rhodamine-labelled albumin, only onto the Si regions of the pattern. In another approach, binary patterns made from silica and amine polystyrene particles are heated at 100 ºC (above glass transition temperature of polystyrene) followed by etching with HF to remove the silica particles creating highly ordered 2- and 3D porous substrates. In summary, we introduce a novel method for generating highly-ordered patterns from colloid crystals that is very fast, inexpensive, and allows patterns of multiple biomolecules over large areas in 2- and 3D.