A lack of healthy transplantable tissue to treat corneal blindness has led researchers to investigate the development and application of different scaffold materials for corneal tissue engineering and regeneration. In this study, hydrogels fabricated from decellularized corneal extracellular matrix were developed as a new approach to corneal stromal tissue regeneration. Porcine corneas were decellularized using a technique that combined freeze-thaw cycles with a nuclease treatment. The corneas were then freeze-dried, milled, and digested in an acidic pepsin solution that was used to form a hydrogel after adjusting the pH and gelation temperature. The resultant corneal matrix hydrogels (CMHs) were seeded with human corneal stromal cells and cultured for several days. When compared to collagens hydrogels, CMHs had superior optical transparency, similar mechanical properties, and were better able to retain the stromal cells native keratocyte phenotype. The CMHs also supported cell viability and proliferation and contained sulfated glycosaminoglycan, a vital constituent, for maintaining corneal transparency. These results suggest that the CMHs could provide an exceptional biomaterial for corneal stroma regeneration.