As a nonlinear optical phenomenon, upconversion (UC) occurs when two or more low-energy excitation photons are sequentially absorbed and emitted. Upconversion nanomaterials exhibit superior photostability, non-invasiveness, a unique near-infrared anti-Stokes shift, and enhanced tissue penetration capability. However, general upconversion nanomaterials typically utilize visible light (400-700 nm) for excitation, leading to limited tissue penetration, background signal interference, limited excitation efficiency and imaging quality issues due to tissue absorption and scattering. The increasing use of upconversion nanomaterials in the near-infrared one-region (NIR-I) window (700-900 nm) offers benefits such as enhanced penetration into biological tissues, relatively improved imaging resolution, and lower spontaneous luminescence, although these materials are still susceptible to background signals, limiting their effectiveness in high signal-to-noise ratio imaging. This distinctive wavelength conversion endows upconversion nanomaterials in the NIR-II region with extraordinary potential for diverse applications. Biomedical research has primarily focused on biomedical imaging for disease diagnosis and treatment, as well as biomarker detection. Nonetheless, studies specifically targeting the NIR-II window remain limited. This paper summarizes the latest research progress on upconversion nanomaterials in the NIR-II region. It begins by introducing the preparation methods for these materials in the NIR-II, followed by their applications in imaging and biological contexts. Lastly, it discusses the primary challenges and future prospects of upconversion materials in NIR-II, aiming to promote their development.