Keratan sulfate (KS) is an important glycosaminoglycan that is found in cartilage, reproductive tissues, and neural tissues. Corneal KS glycosaminoglycan is found N-linked to lumican, keratocan and mimecan proteoglycans, and has been widely studied by investigators interested in corneal development and diseases. Recently, the availability of corneal KS has become severely limited, owing to restrictions on the shipment of bovine central nervous system byproducts across international borders in an effort to prevent additional cases of mad cow disease. We report a simple method for the purification of multi-milligram quantities of bovine corneal KS, and characterize its structural properties. We also examined its protein-binding properties, and discovered that corneal KS bound with high affinity to fibroblast growth factor-2 and sonic hedgehog, a growth factor and a morphogen involved in corneal development and healing. Keratan sulfate prepared in multi-milligram quantities from bovine cornea was structurally characterized by nuclear magnetic resonance spectroscopy and mass spectrometry and the binding of fibroblast growth factors and morphogens was studied using surface plasmon resonance. © 2013 The Authors Journal compilation © 2013 FEBS.