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• A novel ultrasound technique to regenerate flexible protein film without any chemical procedures. • Protein structure, physico-chemical property and biological responses can be tuned by varying ultrasound power. • Mechanism of structural and property changes of silk fibroin films from domestic mulberry and wild tussah under ultrasound is comparative studied. • Tissue bioengineering reuse of natural resource materials. Ultrasonic technology has emerging applications in the advanced biomaterials due to its environmentally friendly and high efficiency. In this work, by scanning electron microscopy, fourier transform infrared spectroscopy, x-ray powder diffraction and thermal analysis techniques as well as hydrophilic and cytocompatible tests, the morphology, structure and physical-biological properties of the silk fibroin (SF) film from Bombyx mori silkworm of Thailand and wild SF from Antheraea pernyi cocoon under 0∼800 W power ultrasonic cavitation were investigated systematically. When the ultrasonic intensity increased from 0 to 800 Mpa, the diameters decreased from 80 to 34 nm for Thailand SF film, and from 110 to 48 nm for tussah film, which exhibited more smooth for the surface of domestic silkworm SF film. It was found that the silk II structure significantly increased after the ultrasonic process, which suggested that the changes of microstructure could provide better thermal stability and mechanical property for regenerated films, such as the ductility of Thailand SF film was more enhanced, whereas the stronger stiffness obtained for Tussash. Besides, the random coils in the domestic SF film were easy to convert into β -sheet structure after ultrasonic treatment, while the wild possessed higher hydrophilic and more cell attachment and proliferation activity due to its abundant (-Ala-) n and (Arg-Gly-Asp) sequence. This comparative study will provide a new strategy of the green design for novel fibroin-based functional materials with tunable structure and properties. [Display omitted] [ABSTRACT FROM AUTHOR]