Background: The aim of the study was to investigate if feto-maternal transfusion was related to the size of the fetal-maternal interface, and, therefore, was larger in twin pregnancy in comparison with singleton pregnancy. Methods: Blood samples from women with singleton (n = 11), and monochorionic (n = 11) and dichorionic (n = 13) twin gestations were tested. Flow cytometry tests with hemoglobin F, glycophorin A, and hemoglobin F and carbonic anhydrase simultaneous staining were used to detect fetal red blood cells and maternal F cells. Results: In all cases, the volume of feto-maternal transfusion was estimated to be low. The highest rate of fetal red blood cells in the maternal circulation was observed in the blood of women with dichorionic twin gestations both before and after delivery. An increase in fetal red blood cells was observed after cesarean section in singletons and twins. The median rate of maternal F cells was 2.23% in singleton, 2.1% in monochorionic and 3.95% in dichorionic pregnancy. Conclusions: Feto-maternal transfusion during pregnancy may be related to the multiplicity and chorionicity of pregnancy.