Background: The role of estrogen and progesterone in stroke is suggested in previous studies, but yet is controversial. Objectives: Comparing the plasma levels of estrogen and progesterone and their ratio in ischemic stroke patients and healthy population. Materials and Methods: This cross-sectional comparative study was conducted from March to September of 2013. Sixty-six female patients older than 60 years with ischemic stroke who referred within the first 12 hour of their neurologic symptoms were included. A total of 48 women who were in the same age range comprised the control group. The serum levels of progesterone and 17-β-estradiol concentrations were measured. The data were analysed by Chi2 test, univariate and logistic regression models in SPSS software 19. Results: There was no significant difference between case and control groups in serum levels of estrogen (28.66 ± 13.60 vs. 35.72 ± 28.36) and progesterone (1.07 ± 0.83 vs. 1.40 ± 1.87) and their ratio (p > 0.05). But in multivariate analysis, estrogen (p = 0.033), progesterone (p = 0.02) and the interaction between them (p = 0.013) remained as associated factors. In addition, the higher ratio of estrogen to progesterone indicates the less ability of patients, which one unit increase in this ratio results in 0.072 reduction in NIHSS (National Institutes of Health Stroke Scale) score. Conclusions: The level of estrogen and progesterone and interaction between them are associated with ischemic stroke. The ratio of estrogen to progesterone has reverse association with score of the ability of patient according to NIHSS.