Abstract Background To explore the relationship between corneal biomechanical parameters and corneal sublayer thickness using corneal visualization Scheimpflug technology (Corvis ST) and ultrahigh-resolution optical coherence tomography (UHR-OCT) in clinical and suspected keratoconus and normal eyes. Methods Cross-sectional prospective study. A total of 94 eyes of 70 participants were recruited. Twenty five eyes of 19 keratoconus patients, 52 eyes of 34 patients showing high risk of developing keratoconus according to the Belin/Ambrosio Enhanced Ectasia Display, and each eye of 17 normal subjects were enrolled. All participants underwent Corvis ST, Pentacam, and UHR-OCT examinations at the same time. Stiffness parameter A1 (SP-A1), deformation amplitude ratio (DA ratio), and other biomechanical parameters were recorded using Corvis ST. The vertical and horizontal thickness profiles of central 3 mm corneal epithelium, Bowman’s layer, and stroma as measured by the perpendicular distance between the neighboring interfaces were generated using UHR-OCT. The flat keratometry and steep keratometry were obtained using Pentacam. Analysis of correlation was applied to explore the association between variables. Results Most of the biomechanical parameters and corneal sublayer thickness profiles showed statistical differences among three groups. A statistically significant linear relationship was noted between SP-A1 and DA ratio in all three groups. SP-A1 was found to be positively correlated with epithelial and Bowman’s layer thickness in the keratoconus (KC) group, and with stromal thickness in all three groups. In the normal and suspected keratoconus (SKC) groups, only stromal thickness was included in the stepwise linear regression to predict SP-A1, whereas in the KC group, steep keratometry and Bowman’s layer thickness were included. Conclusions Significant and different correlations were noted between corneal stiffness and corneal sublayer thickness in different groups, indicating that corneal sublayers may play different roles in maintaining corneal biomechanical stability between keratoconus and normal eyes.