We present an automated method to perform accurate, rapid, and objective measurement of the blood oxygen saturation over each segment of the retinal vascular hierarchy from dual-wavelength fundus images. Its speed and automation (2 s per entire image versus 20 s per segment for manual methods) enables detailed level-by-level measurements over wider areas. An automated tracing algorithm is used to estimate vessel centerlines, thickness, directions, and locations of landmarks such as bifurcations and crossover points. The hierarchical structure of the vascular network is recovered from the trace fragments and landmarks by a novel algorithm. Optical densities (OD) are measured from vascular segments using the minimum reflected intensities inside and outside the vessel. The OD ratio (ODR=OD600/OD570) bears an inverse relationship to systemic HbO2 saturation (SO2). The sensitivity for detecting saturation change when breathing air versus pure oxygen was calculated from the measurements made on six subjects and was found to be 0.0226 ODR units, which is in good agreement with previous manual measurements by the dual-wavelength technique, indicating the validity of the automation. A fully automated system for retinal vessel oximetry would prove useful to achieve early assessments of risk for progression of disease conditions associated with oxygen utilization.