Interest in developing tissue-engineered cornea has increased with the decrease in the supply of donor tissue; however, the high strength and transparency of the cornea present a challenge. Both the collagen processing and crosslinking methods were hypothesized to influence the optical and biomechanical properties of collagen matrices, while regular surface topography was hypothesized to align stromal fibroblasts. Improved transparency and strength were observed when soluble tropocollagen was added to the insoluble collagen and when glucose-mediated ultraviolet (UV) crosslinking as opposed to dehydrothermal crosslinking was used. The fraction of transmittance of the collagen films fabricated from insoluble collagen and soluble tropocollagen and glucose-mediated UV crosslinking was initially 0.91 ± 0.02 and 0.98 ± 0.01 for the smooth films and 0.90 ± 0.02 and 0.97 ± 0.02 for the microgrooved films at 400 and 700 nm and was comparable to that of the native cornea, while the relaxed modulus and ultimate tensile strength ranged from 0.9 to 9.4 MPa and from 0.7 to 4.1 MPa, respectively, over the 3 weeks of culture and were initially at or below the range of values for the native cornea. These collagen scaffolds were significantly stronger and more transparent than previous scaffolds, and aligned stromal fibroblasts were observed on microgrooved surfaces.