During recent decades, a giant leap in the development of nanotechnology has been witnessed. Numerous nanomaterials with different dimensions and unprecedented features have been developed and provided unimaginably wide scope to solve the challenging problems in biomedicine, such as cancer diagnosis and therapy. Recently, two-dimensional (2D) transition metal dichalcogenide (TMDC) nanosheets (NSs), including MoS , WS , and etc., have emerged as novel inorganic graphene analogues and attracted tremendous attention due to their unique structures and distinctive properties, and opened up great opportunities for biomedical applications, including ultrasensitive biosensing, biological imaging, drug delivery, cancer therapy, and antibacterial treatment. A comprehensive overview of different synthetic methods of ultrathin 2D TMDC NSs and their state-of-the-art biomedical applications, especially those that have appeared in the past few years, is presented. At the end of this review, the future opportunities and challenges for 2D TMDC NSs in biomedicine are also discussed.