Background: Corneal collagen cross-linking (CXL) is a procedure for making bonds that connect polymer chains to one another. Corneal CXL aims to slow or stop the progression of keratoconus by using photooxidative therapy so as to increase stromal rigidity. Transglutaminase enzymes are currently widely used in the food industry. Recent studies have shown that mRNA, fibronectin, and transglutaminase were found to be more abundant in human corneal keratocytes treated with UVA and riboflavin. Transglutaminase is considered to reduce discomfort caused by UVA irradiation. Methods: A total of 21 white New Zealand rabbits were divided into three groups, namely, transglutaminase-induced CXL group, epithelial-off CXL group, and transepithelial CXL group. The ocular surface was treated with a 1 U/mL microbial transglutaminase solution, and both the epithelial-off and transepithelial groups were exposed to clinical ultraviolet A-riboflavin (UVA/RF). The efficacy of each group was evaluated on the 14th day after the procedures. The central corneal thickness (CCT) and intraocular pressure (IOP) were evaluated using Corneal Visualization Scheimpflug Technology (Corvis ST). Results: The transglutaminase-induced CXL group exhibited the highest mean CCT (370.14 ± 38.85) in comparison with the UVA/RF epithelial-off group (368.00 ± 25.48) and the UVA/RF transepithelial group (369.86 ± 23.43). The transglutaminase-induced CXL group had the highest IOP mean (8.50 ± 3.02) compared with the UVA/RF epithelial-off (6.50 ± 3.07) and UVA/RF transepithelial groups (7.00 ± 1.90). There were no significant differences in CCT (p = 0.990) or IOP (p = 0.563) between the groups. Conclusions: The findings of this study suggest that there are no significant differences between the transglutaminase-induced CXL group and the UVA/RF CXL group. The safety of transglutaminase-induced CXL could be comparable to that of UVA/RF CXL in terms of altering CCT and IOP, which are two factors contributing to corneal rigidity.