Hyperspectral imaging (HSI) combines spectroscopy and imaging technology, and can acquire multi-dimensional information that includes two-dimensional spatial information and one-dimensional spectral information. The space-resolved spectral images obtained by HSI can provide diagnostic information about histophysiology, morphology, and composition of tissues. HSI is an emerging medical imaging modality and has broad potential applications in the field of biomedicine, especially in disease diagnosis and image-guided surgery. We introduce the basic principles of HSI, the configuration and characteristics of HSI system, and review the recent developments of HSI in biomedical field and its applications in disease diagnosis and surgical guidance. The future research direction and development prospects of HSI are also analyzed and forecasted.