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Abstract Thin polymeric films are being explored for biomedical uses such as drug delivery, biofiltration, biosensors, and tissue regeneration. Of specific interest is the formation of mechanically flexible sheets, which can be formed with controllable thickness for sealing wounds, or as biomimetic cellular constructs. Flexible substrates with precise micro‐ and nanopatterns can function as supports for cell growth with conformal contact at the biointerface. To date, approaches to form free‐standing, thin sheets are limited in the ability to present patterned architectures and micro/nanotextured surfaces. Other materials have a lack of degradability, precluding their application as cellular scaffolds. An approach is suggested using biocompatible and biodegradable films fabricated from silk fibroin. This work presents the fabrication and characterization of flexible, micropatterned, and biodegradable 2D fibroin sheets for cell adhesion and proliferation. A facile and scalable technique using photolithography is shown to fabricate optically transparent, strong, and flexible fibroin substrates with tunable and precise micropatterns over large areas. By controlling the surface architectures, the control of cell adhesion and spreading can be observed. Additionally, the base material is fully degradable via proteolysis. Through mechanical control and directing the adherent cells, it is possible to explore interactions of cells and the microscale geometric topography.