Background <p>There is a significant gender difference in myopia—the incidence of myopia is higher in females than in males. Previous studies have shown that sex hormones may play an important role in the pathogenesis of myopia, but the specific mechanism remains unclear. This study aims to explore the association between testosterone and related biomarkers and the risk of myopia.</p> Methods <p>This study conducted a retrospective case–control study using data from the National Health and Nutrition Examination Survey (NHANES, 1999–2004). A total of 1,280 participants were included (563 in the myopia group and 717 in the non-myopia group), all of whom were male. Three testosterone-related biomarkers were analyzed: total testosterone (TT), sex hormone-binding globulin (SHBG), and free androgen index (FAI). Multivariate logistic regression analysis was used to control for confounding factors such as age, race, education level, body mass index, and income. The non-linear association was evaluated using the restricted cubic spline model. Additionally, the two-sample Mendelian randomization (MR) analysis method was employed to assess the causal effect of bioavailable testosterone on myopia using genetic variations as instrumental variables.</p> Result <p>In the observational analysis, the univariate model indicated a significant inverse association between higher Free Androgen Index (FAI) and the risk of myopia (OR = 0.983, 95% CI 0.973–0.993), while increased Sex Hormone-Binding Globulin (SHBG) levels were associated with an elevated risk of myopia (OR = 1.004, 95% CI 1.0004–1.009). However, these associations were no longer statistically significant after adjusting for confounding factors (all <i>p</i> &gt; 0.05). In the Mendelian randomization analysis, genetically predicted higher levels of bioavailable testosterone showed a causal association with a reduced risk of myopia (IVW method: OR = 0.994, 95% CI 0.989–0.999, <i>p</i> = 0.043). Sensitivity analyses using the weighted median (<i>p</i> = 0.017), simple mode (<i>p</i> = 0.03), and weighted mode (<i>p</i> = 0.042) methods yielded consistent directions of effect. However, the association did not retain statistical significance after multiple testing correction using the Bonferroni method (adjusted threshold = 0.0167).</p> Interpretation <p>These research results indicate that bioavailable testosterone may have a positive effect on preventing myopia, which might explain the higher incidence of myopia among women. However, this relationship failed to maintain statistical significance after multiple comparisons were corrected, suggesting that the results of this study are exploratory findings and require further verification through larger-scale independent cohort studies.</p>