Hormonal transition among middle-aged women with type 2 diabetes may exert an impact in the development of diabetic retinopathy (DR). In this cross-sectional study, we aimed to assess the levels of estradiol, follicle-stimulating hormone (FSH), and luteinizing hormone (LH) in the presence of DR and their relationship with risk factors for DR among pre- and post-menopausal women with type 2 diabetes. Serum levels of estradiol, FSH, and LH were measured using the immunoassay technique. All statistical analysis was performed using SPSS software. From the 255 participants, diabetes duration-matched, 35 premenopausal, and 57 postmenopausal women were selected for analysis. The levels of estradiol, LH, and FSH were found to be similar in participants with and without DR among pre- and post-menopausal women with diabetes. Estradiol level was not found to be related with the risk factors of DR among women with type 2 diabetes. In conclusion, female sex hormone, estradiol is not related to the presence of DR. Further prospective studies are necessary to reveal the mechanistic role of this hormone in the development of DR.