Intrinsic optical signals imaging (IOSI) is a novel technique for functional neuroimaging in vivo, especially in the study of cortical spreading depression (CSD). At 550 nm wavelength, the optical images during CSD showed significant vasodilatation of some small arteries in the surface of cortex of rats. In order to quantify the arteries’ diameter change, two kinds of threshold segmentation methods are applied, one is Isodata algorithm thresholding, the other is Otsu’s thresholding. Firstly, we set up a simple model to prove that segmentation of the vessel in a rectangle region could be equally transferred to describe the diameter change. The two methods could automatically select right thresholds for segmentation, so they were suitable for acquiring the dynamic vasodilatation in a series of optical images by computer. Comparing with the traditional method, the new methods were more robust and with high performance. By the methods, we found the vasodilatation could be distinguished as two processes during one CSD episode, a small vasodilatation before the great one that had been commonly reported before. The hemodynamic character during CSD deserves further study. And the methods can be easily applied to the other optical imaging experiments when the vascular dynamic is concerned.