The main limitation of image processing based on charge-coupled device (CCD) imaging in quantitative evaluation of thin-layer chromatography (TLC) is its nonuniform illumination. This paper presents a novel method to solve the problem. First, the background is estimated by employing the cubic spline surface construction. Then the estimated background is subtracted from the original image to make the image segmentation; Finally, the gray level integration for each object is calculated, which is proportional to the value of samples. Experiment shows that the linear correlation coefficient between gray integration and concentration of samples is 0.9978.