Abstract. Retinal oximetry offers potential for noninvasive assessment of central venous oxyhemoglobin saturation (SO 2 ) via the retinal vessels but requires a calibrated accuracy of ± 3% saturation in order to be clinically useful. Prior oximeter designs have been hampered by poor saturation calibration accuracy. We demonstrate that the bluegreen oximetry (BGO) technique can provide accuracy within ± 3% in swine when multiply scattered light from blood within a retinal vessel is isolated. A noninvasive on-axis scanning retinal oximeter (ROx-3) is constructed that generates a multiwavelength image in the range required for BGO. A field stop in the detection pathway is used in conjunction with an anticonfocal bisecting wire to remove specular vessel reflections and isolate multiply backscattered light from the blood column within a retinal vessel. This design is tested on an enucleated swine eye vessel and a retinal vein in a human volunteer with retinal SO 2 measurements of ∼1 and ∼65%, respectively. These saturations, calculated using the calibration line from earlier work, are internally consistent with a standard error of the mean of ± 2% SO 2 . The absolute measures are well within the expected saturation range for the site (-1 and 63%). This is the first demonstration of noninvasive on-axis BGO retinal oximetry. C 2011 Society of Photo-Optical Instrumentation Engineers (SPIE).