Abstract Photolithography is the most widely used micropatterning technique at the micro‐ and nanoscale in device fabrication. However, traditional photoresists used in photolithography are typically nonaqueous‐based toxic substances that require harsh conditions for processing, limiting the development of biofunctional and biocompatible micropatterns. In this study, a protein‐based aqueous photoresist derived from chemically modified silk fibroin named SAMA, capable of achieving high‐resolution micropatterning (<1.2 µm) while retaining good biocompatibility, is presented. The entire fabrication process, including spin‐coating, development, and lift‐off, employs solely SAMA and water, eliminating the need for toxic reagents and elevated temperature. Notably, the SAMA photoresist allows covalent conjugation of biofunctional molecules, such as enzymes and nucleic acids, while preserving their bioactivity during micropatterning. This innovative approach enables the high‐throughput generation of bioactive micropatterns for various applications such as biosynthesis, diagnostics, and biosensors.