Aim of our study was to evaluate right ventricular (RV) systolic function in neonate using newly developed single-beat three-dimensional echocardiography (sb3DE). We enrolled 15 healthy or premature neonates (0–53 days after birth). We scanned one beat full volume using Siemens ACUSON SC2000 (Siemens AG) echocardiography with 4Z1c full-volume transducer without ECG gating. RV end-diastolic volume (RVEDV) and RV end-systolic volume (RVESV) were computed with special software dedicated to analysis for RV volume. RV ejection fraction (RVEF) and RV stroke volume (3D-RVSV) were calculated. And RV stroke volume was also determined from the recordings of ejection blood flow velocity and diameter at the level of the pulmonary orifice in RV outflow tract (Doppler-RVSV). Tricuspid annular plane systolic excursion (TAPSE) was also measured by 2D echocardiography. RVEDV ranged from 5.1 to 10.7 ml (average 7.5 ml), RVESV ranged from 2.3 to 5.8 ml (average 3.9 ml). There was a good correlation between 3D-RVSV and Doppler-RVSV (r = 0.77). Bland–Altman plot revealed that 3D-RVSV became underestimation of an average of 1.78 ml compared to Doppler-RVSV. And TAPSE positively correlated with 3D-RVEF (r = 0.58, P = 0.038). Newly developed sb3DE enables us to perform three-dimensional acquisition of RV volume without ECG gating even in neonate. However, 3D-RVSV currently tends to be underestimated in neonatal measurement.