The main functions of the conjunctiva, an essential part of the ocular surface, are to maintain the equilibrium of the tear film and to protect the eye. Upon injuries, the prerequisite to successful ocular surface repair is conjunctival reconstruction. Tissue engineering techniques, including transplantation of autografts, amniotic membranes and numerous synthetic/natural materials, have been developed. However, none of these strategies is completely satisfactory due to lack of goblet cell repopulation, poor mechanical properties or non-standardized preparation procedure. Here, we cultured conjunctival epithelial cells on vitrified collagen membranes and developed a tissue equivalent for repairing damaged conjunctiva. Optimized vitrified collagen has superior mechanical and optical properties to previous biomaterials for ocular surface application, and its unique fibrillar structure significantly benefited conjunctival epithelial cell growth and the phenotypic development in vitro. In a rabbit model, vitrified collagen greatly promoted conjunctival regeneration with rapid re-epithelization, sufficient repopulation of goblet cells and minimized fibrosis and wound contracture, proved by gene expression analyses and histological staining. In conclusion, we have demonstrated the potential suitability of utilizing vitrified collagen-based tissue equivalent in ocular surface reconstruction.