The surface structures of ablated rabbit cornea and collagen film were examined using AFM and SEM observations. The surface roughness of the rabbit cornea increased linearly, and then significantly increased as the pulse number increased to 2500. However, the surface roughness of the collagen film increased sharply, and then leveled off as the pulse number increased. Especially, the formation of a conical structure on the dried rabbit cornea and the collagen film was observed, but the formation of the conical structure in situ on the rabbit cornea was not detected. The formation of a conical structure shows that radiation hardening of collagen as a function pulse number has occurred. The presence of hydration in the collagen molecule plays an important role in the surface structure of the cornea during excimer laser ablation.