Micro/nanomanufacturing technology is indispensable for the advanced manufacturing industry, including, but not limited to, microelectronics. Developing novel materials to implement eco-friendly manufacturing with higher performance remains the focus of this field. The creation of ultrafine pattern structures at the nanoscale is enabled by silkworm silk with significantly improved mechanical properties. Here, we report all-water-based manufacturing of nanopattern structures with unprecedented resolution using genetically modified silkworm silk as resists in conjunction with focused ion beam lithography (FIBL), with an average resolution of ~20 nm and a minimum feature size of 11.3 nm. Our study strengthens the outstanding performance of silkworm silk as a natural protein material and presents a promising strategy for manufacturing nanoarchitectures.