Near-infrared afterglow luminescent inorganic nanomaterials (NIR-ALINs) possess the unique property of continuing to emit near-infrared (NIR) luminescence after excitation ceases. They demonstrate excellent photostability, deep tissue penetration, and high imaging signal-to-noise ratio (SNR). Additionally, NIR-ALINs can be re-excited in vivo using visible (Vis), NIR light or X-rays, which avoids the need for continuous in situ excitation, thus eliminating autofluorescence of biological tissues and reducing the tediousness of multiple injections. These features make NIR-ALINs particularly attractive for biological applications. In recent years, a series of NIR-ALINs with prolonged afterglow time and enhanced luminescence intensity have been discovered. However, the development of NIR-ALINs still faces significant challenges, as their NIR afterglow performance is usually insufficient to satisfy practical biological applications. There is still a lack of systematic analysis of the strategies for the regulation of NIR afterglow luminescence in inorganic nanomaterials. This review highlights the rational design and modulation strategies of NIR-ALINs, focusing on host substrate selection, trap engineering modulation and surface modification. Moreover, the biological applications of NIR-ALINs in bioimaging, bio-detection and disease therapy are summarized. Finally, the present challenges and perspectives in biological applications, such as insufficient afterglow properties and unclear biosafety, are also discussed.