There is an increasing demand for new versatile biomaterials. Silk is reported by many researchers as an ideal biomaterial for different biomedical applications. Most of the studies are carried out on mulberry silk Bombyx mori, however silk from Indian non-mulberry silkworms are relatively unexplored. In this report we fabricate 2D matrices from the regenerated aqueous silk fibroin protein of the glands of non-mulberry Indian muga silkworm Antheraea assama (assamensis). Its biochemical, biophysical characteristics and its cytocompatibility for biomedical uses are evaluated. The properties of this muga gland fibroin are compared with silk gland fibroin from non-mulberry Indian tropical tasar silkworm Antheraea mylitta and with the fibroin from the cocoon of mulberry silkworm Bombyx mori. The gland fibroin of Antheraea assama is observed to consist of two polypeptides of approximately 250 kDa each linked by disulfide bond. Fourier transformed infrared spectroscopy and X-ray diffraction studies indicate random coil structure of dissolved fibroin solution. The alpha-helical structure in 2D films changed to beta-sheets upon ethanol treatment, imparting crystallinity and insolubility. The fibroin film is found to be the least hydrophilic, followed by B. mori and A. mylitta silk. Biocompatibility of the films from all three species is investigated through the cell attachment and spreading study of MG-63 human osteoblast-like cells. The cytocompatibility of non-mulberry fibroin matrices are comparable with that of standard tissue culture plates. The results indicate that the non-mulberry Indian muga silk gland fibroin is also suitable matrix as a natural biomaterial for tissue engineering applications.