The ex vivo cultured limbal stem cells over a biocompatible scaffold are used in the management of limbal stem cell deficiency as an ideal replacement for human amniotic membrane (HAM). A novel source of collagen from fish scales (FSC) was used to fabricate the scaffold. In this study, we have evaluated the physicochemical, mechanical, and culture characteristics of FSC and compared with denuded HAM. The cultured corneal cells were characterized by real-time polymerase chain reaction for putative stem cell markers. The swelling ratio, collagenase assay, and microbial resistance of FSC gave better results when compared to those of HAM. The mechanical and physical strengths of FSC were good enough to handle when compared to HAM. Under microscopic observation, epithelial migration was noted at the end of 48 h from limbal explants plated on FSC and on HAM at the end of 72 h. By the end of the 15th day, 90 to 100% confluent growth was seen resembling the morphological features of limbal epithelium. In conclusion, FSCs from a novel renewable biological source were optically clear with sufficient strength, and gave encouraging results in culture studies; the same may be tried as potential candidate for corneal transplantation after in vivo studies.