Fetal lung maturity assessment in twin pregnancy has been discussed, but is still controversial. The purpose of this study is to predict the occurrence of respiratory distress syndrome (RDS) using lamellar body count (LBC) and analyze the validity of LBC for fetal lung maturity assessment in twin pregnancy. Three-hundred two amniotic fluid samples were obtained at cesarean section from 29 to 38 weeks of gestation. Samples were analyzed immediately with no centrifugation and the number of lamellar bodies was counted using a platelet channel on the Sysmex SF-3000. There were 18 neonates (6.0%) suffering from RDS. An LBC cut-off value of 2.95 × 104/μL resulted in 91.5% sensitivity and 83.3% specificity for predicting RDS. This cut-off value for predicting RDS was the same as that in singleton pregnancy. Moreover, the median LBC value in RDS cases was significantly lower than in non-RDS cases (1.50 ± 1.1 × 104/μL vs. 10.6 ± 7.5 × 104/μL; p < .001). This is the first report on the validity of LBC in twin pregnancy and also the largest study on fetal lung maturity assessment in twin pregnancy. An LBC value of >2.95 × 104/μL means reassuring findings for RDS even in twin pregnancy. We believe the data in this study provide valuable, new information for the management of twin pregnancies.