Background: Congenital heart defects are the most common of birth defect, which leads to neonatal death after birth. Early diagnosis during prenatal period would be a benefit for precaution and treatment. Antegrade Late Diastolic Arterial blood Flow (ALDAF) was reported to measure fetal atrioventricular (AV) time intervals (FAVTI) at an early gestational ages (GA) of 6 weeks. There has been no previous studies reporting reference value of fetal atrioventricular time intervals (FAVTI) derive from ALDAF technique. Methods: Using fetal echocardiogram, this cross-sectional study was performed on 528 healthy fetuses between 14 and 40 weeks. Pulsed wave Doppler-derived FAVTI (milliseconds) were measured from ALDAF-AO and ALDAF-PA and left ventricle (LV) In/Out. Correlations between these three Doppler measurement techniques were examined with the Bland-Altman analysis and Pearson correlation coefficient. GA was used as specific reference value and its correlation with FAVTI was examined with linear regression. Results: We establish reference values of fetal atrioventricular (AV) time intervals (FAVTI) from antegrade late diastolic arterial blood flow (ALDAF) aorta (AO) and pulmonary artery (PA) from 14 to 40 weeks of gestation (GA). A positive correlation between FAVTI and GA was identified when using each of the three measurements (ALDAF-AO/ALDAF-PA and LV In/Out) (R2 = 0.177–0.272; P 0.001). GA had the strongest impact on ALDAF-AO FAVTI, which was estimated to have a predicted FAVTI of 1.02 \times GA (weeks) + 87.82. Bland-Altman analysis showed FAVTI of ALDAF-AO and ALDAF-PA were also significantly correlated (R2 = 0.573, P 0.001). Intra-observer and inter-observer reliability coefficients showed good reproducibility (ICC >0.90) for all methods. Conclusions: This is the first study to establish reference ranges for FAVTI obtained from ALDAF-AO/ALDAF-PA for each week of gestation from 14 to 40 weeks. Our findings inform clinical practice by establishing GA-specific ALDAF-AO/PA cut-off values for the diagnosis of congenital heart block. FAVTI from ALDAF-AO/ALDAF-PA is a more practical measurement to use in the clinical setting because it is easier to investigate than LV In/Out. Good reproducibility in FAVTI measurements and a lack of fetal heart rate influence underpin the strength of our findings.