The study comprises 889 pregnant women between 14 and 21 weeks of gestation. The control group consisted of 862 pregant women with unburdened obstetric anamnesis with an uneventful singleton pregnancy. The examined group consisted of 27 pregnant women with uncomplicated twin pregnancy. In the sera of pregant women AFP (Microparticle Enzyme Immunoassay AxSYM Abbott), total (β-hCG (Microparticle Enzyme Immunoassay AxSYM Abbott) and unconjugated estriol (Radioimmunoassay Amerlex-M. 2T Johnson & Johnson Ortho Clinical Diagnostics Ltd.) were determined. The risk of fetal trisomy 21 was calculated with the use of PRISCA 3.0 software, which corrected the MoM values for twin pregnancy. Ulm Index was also calculated. In the majority of twin pregnancies increased concentrations of AFP, total (β-hCG and uE3 in the range over 1,0 MoM was noted. In the group of women below 35 years of age with singleton pregnancies using PRISCA 3.0 software it approximated to 95%. For women older than 35 optimum index for fetal trisomy 21 risk calculation was Ulm Index with the specificity 93,8%. The specificity of AFP determination in the detection of fetal open NTD in singleton pregnancy was 99%. In the group of women with twin pregnancy the obtained specificity of 77,8% for PRISCA 3.0 software is low, a more adavantageous way to calculate the risk of fetal trisomy 21 is Ulm Index with the specificity of 85,2%. The specificity of AFP determination as a screening for fetal open NTD in twin pregnancy was 96,3%.