Biocompatible and biodegradable silk fibroin films show promise as an eco-friendly biomaterial with excellent mechanical, thermal, and optical transparency properties. In contrast, polyimide (PI) films adopted in the liquid-crystal display (LCD) industry for aligning LC molecules are synthesized using toxic chemicals, which are nonrecyclable and nonbiodegradable. In this work, Bombyx mori silk fibroin films are fabricated from the aqueous solution and applied as alignment films for LCDs. The thermal properties of the prepared regenerated silk fibroin materials under different heat treatment temperatures are investigated using differential scanning calorimetry (DSC) and thermogravimetric analysis (TGA). The crystallinity of different heat-treated silk fibroin materials is determined by infrared spectroscopy. The silk fibroin film treated at a higher temperature exhibits better thermal stability due to the higher crystallinity of the β-form structure. The LCDs using silk fibroin alignment films show a low pretilt angle of 0.5° and an anchoring energy of ∼10–3 J/m2 similar to those of the conventional polyimide films.