Second near-infrared (NIR-II) fluorescence imaging has emerged as a breakthrough technology for accurately revealing complex mechanisms in vivo owing to its high sensitivity, deeper tissue penetration, high spatiotemporal resolution, and high throughput. This review provides a comprehensive overview of NIR-II fluorescence imaging, specifically focusing on the materials used, including single-walled carbon nanotubes (SWCNTs), quantum dots (QDs), rare-earth nanoparticles (RENPs), and organic fluorophores (OFs). It details their development, application, and advantageous performance in NIR-II fluorescence imaging. Furthermore, this review highlights an approach to dynamic multiplexed NIR-II fluorescence imaging in vivo that enables multitarget detection, providing a powerful tool for accurately and effectively assessing pathological processes and revealing complex biological mechanisms in vivo. Finally, it explores the aspects of translational medicine for NIR-II imaging, addressing challenges, and future prospects related to material development, detection equipment, and unmet biomedical applications.