Silk fibroin with sophisticated hierarchical architectures from nano to macro scale shows excellent mechanical properties, good biocompatibility, and outstanding processability. In particular, the crystalline region in silk fibroin contributes high strength and toughness. However, it is difficult to obtain the crystalline silk nanofibrils or nanosheets through top-down methods. The existing silk-derived components are mainly amorphous and sacrifice the delicate structure of the pristine silk. Herein, we report a gentle method to extract the crystalline silk nanosheet (SNS) from the degummed silk fibers. The crystalline SNS has seven strands of β-sheet nanocrystal layer and shows a thickness of 2.75 nm. It can assemble into a membrane via a vacuum filtration process and shows high transparency, excellent thermal stability, outstanding cytocompatibility, and efficient dye interception. Moreover, without external stimuli, the crystalline SNS is capable of reversibly self-assembling to well-organized microfibers. The crystalline SNS is not only a new member of silk fibroin derivatives, but also a promising assemblable unit for versatile applications. We anticipate this work will provide a new insight into the construction and applications of diverse two-dimensional (2D) functional silk materials.