The aim of this study was to evaluate the efficacy and safety of trehalose-treated freeze-dried amniotic membrane (TT-FDAM) for ocular surface reconstruction. Human AM deprived of amniotic epithelial cells was first incubated with 10% trehalose solution, and then freeze-dried, vacuum-packed, and sterilized with gamma-irradiation. The resultant newly developed TT-FDAM was characterized for its physical, biological, and morphological properties by comprehensive physical assays, immunohistochemistry, electron microscopy, cell adhesion assay, 3D cell culture, and an in vivo biocompatibility test. The adaptability of TT-FDAM was markedly improved as compared to FDAM. Immunohistochemistry for several extracellular matrix molecules revealed that the process of freeze-drying and irradiation apparently did not affect its biological properties, however, electron microscopy revealed that the detailed morphological appearance of TT-FDAM is more similar to that of native AM than to FDAM. Intracorneal and scleral-surface transplantation of TT-FDAM showed excellent biocompatibility with ocular surface tissues. Thus, TT-FDAM retained most of the physical, biological, and morphological characteristics of native AM, consequently it is a useful biomaterial for ocular surface reconstruction.