Accurate assessment of the fetal organ volumes is very important in the evaluation of fetal well-being and maturation. Previous investigators have pointed out that fetal thigh volume (ThiV) may be a useful predictive factor of intrauterine growth retardation. Yet, 2-D ultrasound (US) is limited in assessing fetal ThiV accurately. With the recent advance of 3-D US, the limitation in assessing fetal ThiV by 2-D US can be overcome. To establish a normal reference chart of fetal ThiV for clinical use, a prospective and cross-sectional study using 3-D US was undertaken to assess the fetal ThiV in normal pregnancy. In total, 204 singleton fetuses ranging between 20 and 40 weeks of gestation and fitting the criteria of normal pregnancies were enrolled in this study. Our results showed that fetal ThiV is highly correlated with the gestational age (GA). Furthermore, using GA as the independent variable and ThiV as the dependent variable, the best-fit regression equation was ThiV (mL) = 35.494 − 4.985 × GA + 0.183 × GA 2 (r = 0.91, n = 204, p < 0.0001). For further clinical use, a chart of normal growth centiles of fetal ThiV was then calculated based on this equation. Furthermore, common indexes of fetal biometry, such as biparietal diameter (BPD), occipitofrontal diameter (OFD), head circumference (HC), abdominal circumference (AC), femur length (FL) and estimated fetal weight (EFW), were all highly correlated with ThiV (all p < 0.0001). In conclusion, our data of fetal ThiV assessed by 3-D US can serve as a useful reference in evaluating fetal growth and nutrition status during normal gestation.