Due to their unique physicochemical characteristics, 2D materials have attracted more and more attention in the biomedicine field. Currently, 2D materials-based nanomedicines have been extensively applied in various diseases including cancer, bacterial infection, tissue engineering, biological protection, neurodegenerative diseases, and cardiovascular disease. Depending on their various characteristics, these 2D nanomedicines exert their therapeutic effect in different ways, showing great clinical application prospects. Herein, we focus on the various biomedical applications of 2D materials-based nanomedicine. The structures and characteristics of several typical 2D nanomaterials with different configurations and their corresponding biomedical applications are first introduced. Then, the potential of 2D nanomedicines on therapeutic and imaging and their biological functionalization are discussed. Furthermore, the therapeutic potentials of 2D nanomedicines in various diseases are also comprehensively summarized. At last, the challenges and perspectives for the advancement of 2D nanomedicines in clinical transformation are outlooks.