Confocal laser scanning microscopy (CLSM)，through the use of fluorescent probes for labeling, enables the visualization of the spatial distribution of components in whole grain foods and the in situ characterization of their microstructure, and has become a core analytical tool in the fundamental research of whole grain processing. However, the current academic community has yet to provide a comprehensive and systematic review of the research progress of this technology in the field of whole grain foods. This paper reviews the preparation methods for whole-grain samples, the multi-component changes and action mechanisms during whole-grain food processing revealed by confocal laser scanning microscopy, and the investigation into the regulatory mechanism of starch digestion influenced by the multi-scale structural barriers in whole grains. Additionally, this paper provides a comprehensive analysis of the current application status and key achievements of CLSM in whole-grain food. Furthermore,it researchs, aiming to offer detailed theoretical references for advancing scientific research and technological innovation in whole-grain foods. Furthermore, it contributes to the implementation of the scientific and technological innovation objectives outlined in the National Whole Grain Action Plan (2024-2035), and promote the high-quality development of the whole-grain food industry.