<p>This study aimed to investigate the relationship between myopia and retinal vascular oxygen saturation, after accounting for optical factors and individual variations. This was a prospective, observational, single-center study. Adults with anisometropia were included. All the participants underwent femtosecond-assisted laser in situ keratomileusis (FS-LASIK), and before and after the surgery, all the patients underwent the examination of visual acuity, intraocular pressure (IOP), spherical equivalent (SE), average keratometry (K), central corneal thickness, and retinal oximeter. A total of 196 adult patients with myopic anisometropia were enrolled. The SE of the less myopic eyes before surgery was − 3.89 ± 3.24 D, which was significantly higher than that of the more myopic eyes − 6.75 ± 3.10 D (<i>p</i> &lt; 0.001). The retinal arterial oxygen saturation (SaO₂) of the less myopic eyes was 94.08 ± 1.61%, which was significantly higher than that of the more myopic eyes 93.36 ± 1.74% (<i>p</i> = 0.003). After FS-LASIK, the SaO₂ of the less myopic eyes (93.29 ± 1.72%) remained significantly higher than that of the more myopic eyes (92.76 ± 1.83%) (<i>p</i> = 0.038). In the multivariate regression analysis, the postoperative SaO₂ was still significantly negatively correlated with axial length (AL) (<i>B</i> = -0.252, <i>P</i> = 0.009). After excluding optical factors and individual variations, SaO₂ remains negatively correlated with AL. Myopia exerts an optical magnification effect on SaO₂.</p>