AbstractSelf‐assembly of a recombinant spider silk protein into nanofibrillar networks in combination with photolithography is used to produce diversely functionalized micropattern. Amino‐modified substrates coated with a positive tone photoresist are processed into 1 µm deep arbitrarily shaped microwells, at the bottom of which spider silk proteins are covalently coupled to the deprotected aminated surface. The protein layer serves to seed the self‐assembly of nanofibrils from the same protein in the microwells, forming immobilized few nanometers thin networks after the stripping of the photoresist. The nanofibrous micropattern can be functionalized by employing fluorescently modified spider silk variants during the self‐assembly or by later covalent modification with nucleic acids. By repeating the photolithography and fibril assembly procedures, two functionally different and spatially defined pattern are created.