Currently, microscopic imaging methods based on fluorescent labeling are the mainstream approaches in biomedical research. However, fluorescent labeling faces bottlenecks, such as photobleaching, photo quenching, specific labeling difficulty, and fluorescence disturbance. Therefore, the development of label-free microscopic imaging techniques has attracted numerous attentions. In this review, four label-free optical microscopy techniques are described: coherent Raman scattering imaging, photothermal imaging, surface plasmon resonance imaging, and interference scattering imaging. The basic principles of these techniques are illustrated, and their applications in biomedical fields, including morphology of biomolecules, viruses, and cells, as well as biodynamics, are described. Finally, the performance of the four imaging techniques is compared, and the advantages, limitations, and application prospects are summarized.