We aimed to investigate the association between serum cortisol, testosterone, and cortisol/testosterone (C/T) ratio and stroke. A total of 6157 subjects were included in this study. The serum levels of cortisol and testosterone were determined by liquid chromatography-tandem mass spectrometry method. The relationships of serum cortisol, testosterone, and C/T ratio levels with the odds ratios (ORs) of stroke were calculated using logistic regression analysis. The dose-response associations between serum cortisol, testosterone, and C/T ratio levels with stroke estimated by restricted cubic splines. After adjusting for multiple variables, serum testosterone levels were negactively associated with stroke in males (per 1 unit, odds ratio (OR) = 0.83, 95 % confidence interval (CI): 0.70, 0.97). The serum C/T ratio is positively associated with stroke in both males and females (OR = 1.16, 95 % CI:1.04,1.34 in males and OR = 1.15, 95 % CI:1.02,1.31 in females). Restricted cubic splines showed a significant linear dose-response relationship between testosterone and C/T ratio and stroke in males (P for overall association <0.05, P for nonlinearity >0.05). Our study findings show that serum testosterone levels are negatively correlated with stroke in males; meanwhile, serum C/T ratio is positively correlated with stroke in both males and females.