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To determine whether late second-trimester maternal serum biomarkers are useful for the prediction of preeclampsia during the third trimester, a case-control study including 33 preeclamptic and 71 healthy pregnancies was conducted. Maternal serum concentrations of placental protein 13 (PP13), pregnancy-associated plasma protein (PAPP-A), pentraxin3 (PTX3), soluble FMS-like tyrosine kinase-1 (sFlt-1), myostatin and follistatin-like-3 (FSLT-3) were measured at 24-28 weeks’ gestation. All the concentrations of these markers were compared between the preeclamptic and control groups. Receiver operating characteristic (ROC) curve analysis was applied to assess sensitivity and specificity of serum markers with significant difference.The levels of PP13 and sFlt-1 were significantly increased and FSLT3 was significantly decreased in patients with preeclampsia. However, the concentration of PAPPA, PTX3 and myostatin did not differ significantly. In screening for preeclampsia during the third trimester by PP13, sFlt-1 and FSLT3, the detection rate was 61.3%, 48.1% and 39.1%, respectively, at 80% specificity, and the detection rate increased to 69.8% by combination of these three markers.Maternal serum levels of PP13, sFlt-1 and FSLT3 play an important role in predicting late-onset preeclampsia, and the combination of these three markers significantly increases the detection rate for prediction.