Optical imaging, with its advantages of being radiation-free, simple to operate, high spatiotemporal resolution, and real-time imaging, is widely used in the life sciences. Among these, short-wave infrared (SWIR) imaging technology is a recent emerging research hotspot in optical imaging, and its significant potential in in vivo animal biological function research and clinical translation has expanded the medical research and clinical applications of optical imaging. This article systematically reviews the progress of SWIR imaging applications in in vivo animals, including cellular-level tracing, real-time dynamic visualization of the vascular system, and dynamic monitoring of specific pathophysiological processes. It also focuses on the recent clinical translational advancements of SWIR imaging, particularly its advantages and potential in surgical navigation applications. With the continuous optimization and development of contrast agents (such as fluorescent probes) and imaging equipment (such as multimodal imaging), SWIR imaging holds immense potential for future applications in precision medicine research and clinical diagnosis and treatment.