Abstract: One of the major problems in obstetrics and pediatrics is preterm birth. A new method of prediction of preterm birth is by salivary estriol. This study aimed to determine the predictive value of single measurement of salivary estriol and its relationship with preterm birth. In this study, the salivary specimens of 466 pregnant women of 25-34 weeks gestational age were collected and kept in a freezer until delivery. Consequently, the salivary specimens were thawed and estriol levels were measured. The cut-off point for estriol was determined by a receiver operating characteristics curve. Salivary estriol levels equal to or higher than the cut-off point (2.6 ng mL-1) were considered as the estriol (+) group and those lower than 2.6 ng mL-1 were considered as the estriol (-) group. Our findings showed that 36 (18.3%) subjects in the estriol (+) group and 22 (8.2%) subjects in the estriol (-) group had preterm deliveries. There was a significant relationship between salivary estriol levels and preterm birth (χ2 = 10.636, p = 0.001). Sensitivity, specificity and predictive values (positive and negative) of estriol were 62, 60, 18.3 and 82%, respectively. Single measurement of salivary estriol at 25-34 weeks of gestation, with its high negative predictive values, could be beneficial to identify women who will not develop preterm labor. This outcome suggests that unnecessary interventions should be avoided to prevent preterm births.