Cornea tissue engineering is strictly dependent on the development of biomaterials that fulfill the strict biocompatibility, biomechanical, and optical requirements of this organ. In this work, we generated novel biomaterials from the squid gladius (SG), and their application in cornea tissue engineering was evaluated. Results revealed that the native SG (N-SG) was biocompatible in laboratory animals, although a local inflammatory reaction was driven by the material. Cellularized biomaterials (C-SG) demonstrated that the SG provides an adequate substrate for cell attachment and growth, and corneal epithelial cells cultured on this biomaterial were able to express crystallin alpha, a marker for this type of cells. Biomechanical analyses showed that N-SG biomaterials have higher Young modulus and lower traction deformation than control native corneas (CTR), and C-SG showed a similar Young modulus than CTR. Analysis of the optical properties of these samples revealed that the diffuse transmittance of N-SG and C-SG were higher than CTR, with the diffuse reflectance showing the opposite behavior. These results confirm the putative usefulness of this abundant marine-derived biomaterial that can be obtained as a byproduct of the fishing industry.