Quantitative analysis of molecular structural change by means of Diffracted X-ray tracking (DXT) method requires tracking of dozens of bright spots in noisy image sequence. As the observation equipment upgrades, experimental data was cannot be analyzed manually with sufficient speed and accuracy. We propose automatic analysis method combining background estimation and frame-by-frame matching. We applied propose method to real DXT image data acquired for potassium ion channel structural changing analysis for evaluation. The result of experiments show that the method has high accuracy and be able to replace manual analysis.