Silk fibroin (SF) is a natural material with polymorphic structures that determine its water solubility and biodegradability, which can be altered by exposing it to heat. Here, a hybrid thermal lithography method combining scalable microscale laser-based patterning with nanoscale patterning based on thermal scanning probe lithography is developed. The latter enables in addition grayscale patterns to be made. The resolution limit of the writing in silk fibroin is studied by using a nanoscale heat source from a scanned nanoprobe. The heat thereby induces local water solubility change in the film, which can subsequently be developed in deionized water. Nanopatterns and grayscale patterns down to 50 nm lateral resolution are successfully written in the silk fibroin that behaves like a positive tone resist. The resulting patterned silk fibroin is then applied as a mask for dry etching of SiO2 to form a hard mask for further nano-processing. A very high selectivity of 42:1 between SiO2 and silk fibroin is obtained allowing for high-aspect ratio structure to be fabricated. The fabricated nanostructures have very low line edge roughness of 5 ± 2 nm. The results demonstrate the potential of silk fibroin as a water-soluble resist for hybrid thermal lithography and precise micro/nanofabrication.