Three-dimensional quantitative coronary angiography (3D QCA) has been encouraged by the increasing need to better assess vessel dimensions and geometry for interventional purposes. A novel 3D QCA system based on biplane X-ray angiograms is presented in this paper. By correcting for the isocenter offset and by improving the epipolar constraint for corresponding two angiographic projections, accurate and robust reconstruction of the vessel centerline is achieved and the reproducibility of its applications, e.g., the assessments of obstruction length and optimal viewing angle, is guaranteed. The accuracy and variability in assessing the obstruction length and optimal bifurcation viewing angle were investigated by using phantom experiments. The segment length assessed by 3D QCA correlated well with the true wire segment length (r (2) = 0.999) and the accuracy and precision were 0.04 +/- 0.25 mm (P < 0.01). 3D QCA slightly underestimated the rotation angle (difference: -1.5 degrees +/- 3.6 degrees , P < 0.01), while no significant difference was observed for the angulation angle (difference: -0.2 degrees +/- 2.4 degrees , P = 0.54). In conclusion, the new 3D QCA approach allows highly accurate and precise assessments of obstruction length and optimal viewing angle from X-ray angiography.