The objective of this study was to identify the predictive value for preeclampsia of second-trimester serum high mobility group box-1 (HMGB1) and uterine artery Doppler in singleton pregnancies. Between April 2020 and April 2021, a prospective study was conducted on singleton pregnancies with a gestational age of 16–20+6 weeks at King Chulalongkorn Memorial Hospital, Department of Obstetrics and Gynecology, Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand. Maternal characteristics, uterine artery Doppler, and serum HMGB1 were collected. Serum HMGB1 levels and mean uterine artery pulsatility index (UAPI) were combined to calculate the predictive value for preeclampsia. A total of 393 pregnant women were analyzed, with 25 cases (6.4%) developing preeclampsia and 5 cases (1.3%) developing early-onset preeclampsia. Baseline characteristics of preeclampsia and normal pregnant women were comparable. Preeclamptic pregnant women had significantly higher mean serum HMGB1 levels than normal pregnant women (1112.8 ± 363.1 ng/mL vs 910.8 ± 486.1 ng/mL, p = 0.013). There was no difference in the mean UAPI. Any early-diastolic notching was found more frequently in the preeclampsia group (32.0% vs 12.5%, p = 0.013). The cut-off value for serum HMGB1 levels above 1.04 MoM as abnormal value to predict preeclampsia had sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of 88.0%, 53.5%, 11.4% and 98.5%, respectively. When using abnormal serum HMGB1 levels combined with mean UAPI above 95th percentile, the sensitivity, specificity, PPV and NPV to predict preeclampsia were 88.0%, 50.8%, 10.8% and 98.4%, respectively. This study showed that serum HMGB1 at 16–20+6 weeks of gestation were effective in predicting preeclampsia. The addition of UAPI did not improve the prediction performance.