In this paper, we propose a creative non-contact automatic optical inspection (AOI) method for slit Mura measurements using a spectrometer and a photomultiplier tube (PMT). The experimental results indicate significant breakthrough of uniformity detection than typical macro CCD-based sensors and naked eyes, even the color difference is below the Just Noticeable Difference (JND) level. The characteristic of optical density (OD) is tended to be a concave curve; by contrast to chromaticity profile which shows a convex feature. Using the feature of chromaticity or optical density profiles across a slit Mura, the color difference from 0.12% to 2.71% in CIEy can be measured accurately. OD difference from 0.01 to 1.11 can be detected as well. Among of the samples, the most significant change is the 2.71% of CIEy chromatic variation versus a 1.1 OD difference along the slit Mura which is explored to quantity the JND's perceptibility. A higher chromaticity corresponds to a higher transmittance and a lower optical density, and can be deduced accurately by the present method.