The distribution and transfer behavior of fibers within yarns has garnered significant research interest. Synchrotron radiation three-dimensional (3D) micro X-ray computed tomography (μ-XCT) is known for its strong penetration and high resolution and is used to visualize the internal structures of objects. In this study, a cotton yarn (ring-spun cotton yarn) was scanned, then reconstructed using 3D printing, visualized, and trimmed using the AI-assisted segmentation module in the Avizo software. The results showed that the sub-micrometer-resolution 3D microscopic imaging of cotton yarn could be achieved. This can be used to acquire the structural features of the yarn and its fiber transfer characteristics. A comprehensive understanding of the structure and performance of yarns was developed, providing valuable information for optimizing cotton yarn production and improving yarn quality and performance.