Abstract. The aim of the present study was to evaluate the serum levels of α-fetoprotein (AFP), free-β-human chorionic gonadotropin (free-β-hCG) and unconjugated estriol (uE 3 ) in second trimester maternal serum and comparative analysis of serum markers levels in normal twin and singleton pregnancies, and to evaluate the feasible methods for the screening performances of triple markers in twin pregnancy. The levels of maternal serum markers were measured by magnetic microparticle chemiluminescence immunoassay in the second trimester. TCSoft was used for calculating the risk of Down's syndrome (DS). The marker levels were compared between the twin and singleton pregnancies, along with gestational age, and the weight or age of the pregnant women. The concentrations of the maternal serum markers were higher on average in twin compared to in singleton pregnancies in the second trimester of the gestation. The levels of AFP were 2-fold higher in twin compared to those in singleton pregnancies in weeks 16, 17 and total gestational weeks. For free-β-hCG, the levels were higher in 15 and 18 gestational weeks. However, significant differences were found in all the gestational weeks for uE 3 . There was no correlation between the levels of markers and weight or age in the twin pregnancies. The serum marker levels of AFP, free-β-hCG and uE 3 in normal twins were not all 2-fold higher compared to singleton pregnancies. The current singleton gestational age-specific model for DS screening requires further correction so that it is feasible for screening in twin pregnancies.