Optical tweezers, which leverage the forces exerted by radiation pressure, have emerged as a pivotal technique for precisely manipulating and analyzing microscopic particles. Since Arthur Ashkin's ground-breaking work in the 1970s and the subsequent development of the single-beam optical trap in 1986, the capabilities of optical tweezers have expanded significantly, enabling the intricate manipulation of biological specimens at the micro- and nanoscale. This review elucidates the foundational principles of optical trapping and their extensive applications in the biomedical sciences. The applications of optical tweezers in biomedicine are vast, ranging from the investigation of cellular mechanical properties, such as cell stretching, membrane elasticity, and stiffness, to single-molecule studies encompassing DNA and protein mechanics, protein-DNA interactions, molecular motor functions, and pathogen-host interactions. Advancement of optical tweezers in this field includes their integration with holography, fluorescence microscopy, microfluidics, and enhancements in force sensitivity and positional accuracy. These tools have profoundly impacted the study of cellular mechanics, drug discovery processes, and disease diagnostics, providing unparalleled insights into the biophysical mechanisms underlying health and pathology.