Biologically relevant nanopatterns are useful platforms to address fundamental questions, for example, regarding protein–protein and cell–protein interactions. For the creation of nanopatterns, complex and expensive instrumentation is often needed. We present a simple but versatile patterning method using a combination of particle and subsequent molecular self-assembly to produce ordered structures in the micron and sub-micron range. Polystyrene particles were, in a first step, assembled via dip-coating or dried in a drying cell. Silicon wafers and glass slides coated with SiO2 and a top layer of 11 nm of TiO2 were used as substrates. Large hexagonally ordered particle monolayers were formed with high reproducibility. These were subsequently shrunk in a controlled manner by exposure to a O2/N2 plasma and subsequently used as etching masks to transfer the particle pattern onto the substrate, creating TiO2 features in an SiO2 background. After removing the mask the oxide contrast was translated in three simple dip-and-rinse steps into a biochemical contrast of protein-coated features in an inert background. In short, alkane phosphates were first selectively adsorbed to the TiO2 features. Then the SiO2 background was backfilled using poly(L-lysine)–graft-poly(ethylene glycol) and finally streptavidin was adsorbed to the hydrophobic alkane phosphate SAMs, allowing subsequent binding and hybridization of biotinylated DNA.