We present a novel artery-vein separation method using 0.1-Hz oscillation at two wavelengths with optical imaging of intrinsic signals (OIS). The 0.1-Hz oscillation at a green light wavelength of 546 nm exhibits greater amplitude in arteries than in veins and is primarily caused by vasomotion, whereas the 0.1-Hz oscillation at a red light wavelength of 630 nm exhibits greater amplitude in veins than in arteries and is primarily caused by changes of deoxyhemoglobin concentration. This spectral feature enables cortical arteries and veins to be segmented independently. The arteries can be segmented on the 0.1-Hz amplitude image at 546 nm using matched filters of a modified dual Gaussian model combining with a single Gaussian model. The veins are a combination of vessels segmented on both amplitude images at the two wavelengths using multiscale matched filters of single Gaussian model. Our method can separate most of the thin arteries and veins from each other, especially the thin arteries with low contrast in raw gray images. In vivo OIS experiments demonstrate the separation ability of the 0.1-Hz based segmentation method in cerebral cortex of eight rats. Two validation studies were undertaken to evaluate the performance of the method by quantifying the arterial and venous length based on a reference standard. The results indicate that our 0.1-Hz method is very effective in separating both large and thin arteries and veins regardless of vessel crossover or overlapping to great extent in comparison with previous methods.