Background: Gestational diabetes is the most common antenatal medical complication that is associated with adverse short-and long-term maternal, fetal, and neonatal outcomes. Reducing maternal and fetal complications requires the early diagnosis of gestational diabetes. Unconjugated Estriol (UE) has led to insulin resistance under in vitro conditions. Objectives: This study aimed to determine the predictive power of unconjugated estriol in the diagnosis of gestational diabetes in Tehran, Iran. Methods: The present historical cohort study was conducted on 523 pregnant women presenting to two university-affiliated hospitals in Tehran, Iran, 2017-2018. The level of unconjugated estriol was determined at the 14th-17th week of pregnancy, and gestational diabetes was diagnosed at the 24th-28th week of pregnancy using the oral glucose tolerance test with 75 grams of glucose. Data were collected through interviews and sampling was carried out using a convenience sampling method. Results: Out of 523 pregnant women examined, 63 (12%) were placed in the gestational diabetes group and 460 (88%) in the nongestational diabetes group. The best cutoff point for unconjugated estriol was determined using the ROC curve as 0.965 MOM. We obtained 66.66% sensitivity, 54.78% specificity, 16.8% positive predictive value, and 92.30 negative predictive value for the UE test. Conclusions: Given the acceptable sensitivity (66.66%) and specificity (54.78%) obtained for the UE test and the area under the ROC curve of 0.60, it appears that the UE test can be considered a new, accessible, and reliable screening test for gestational diabetes.