© 2021, The Author(s), under exclusive licence to Springer Nature B.V. part of Springer Nature.Purpose: To assess the activity of xanthine oxidase (XO) enzyme in keratoconic corneal epithelium and to evaluate its relationship with the keratoconus (KC) severity. Methods: This prospective and randomized study included 66 eyes of 54 KC patients who received corneal collagen cross-linking treatment and 43 eyes of 32 patients who underwent photorefractive keratectomy due to their refractive error. During surgical procedures, the corneal epithelium was mechanically scraped and gathered to analyze the XO enzyme activity spectrophotometrically. The KC group was subdivided into three groups (stages 1, 2, and 3) according to the Amsler–Krumeich classification. The results were compared between the KC and the control group and in between KC subgroups. Results: No significant differences in age and gender were found between the KC and control groups (p = 0.064 and p = 0.296, respectively). The mean XO activity levels of the KC and control groups were 173.57 ± 87.61 and 223.70 ± 99.52 mIU/mg, respectively (p < 0.001). In KC group, 33 eyes were at stage 1, 19 were at stage 2, and 14 were at stage 3. No significant difference was observed between KC subgroups regarding XO activity levels (p = 0.681). Conclusion: In this study, our findings revealed that ultraviolet-related pro-oxidant XO enzyme may have a role in the etiopathogenesis of KC. Further studies are needed to support our result. Clinical Trials Registration: When we started this study in 2018, we did not have a "Clinical Trials Registration." However, we have ethics committee approval (date: 21. 02. 2018/No: 22).