With the continuous development of nanotechnology, nanoformulations show unique advantages in improving drug delivery and bioavailability. However, most nanocarriers have low drug delivery efficiency, poor therapeutic effect, potential systemic toxicity and metabolic instability. In recent years, self-assembled carrier-free nanodrugs have attracted tremendous attentions in the field of biomedicine due to their unique properties such as high drug loading capacity, low toxicity, and facile synthesis. Therefore, the self-assembled carrier-free nanodrugs exhibit broad application prospects and development potential in biomedical fields, especially in anticancer and antibacterial applications. In this review, we firstly give a brief introduction to the various intermolecular interactions of self-assembly carrier-free nanodrugs, including the hydrogen bonding, π-π stacking, hydrophobic interaction and other non-covalent forces as exemplified by electrostatic interaction and Van der Waals forces. The chemical structures of drug molecules determine the strength of non-covalent interactions. Secondly, we provide an overview of the typical methods used for self-assembly of carrier-free nanodrugs including in vitro self-assembly strategy (e.g., top-down, anti-solvent precipitation, template-assisted precipitation) and in vivo self-assembly strategy. Especially, nanodrugs prepared by in vivo self-assembly method can be targeted and self-assembled at the target location, reducing adverse reactions and achieving higher efficacy. Besides, the application of carrier-free nanodrugs in biomedical fields including anticancer, antibacterial, anti-inflammatory as well as antioxidant are comprehensively reviewed. Finally, the future challenges and development trends of carrier-free nanodrugs are also prospected, which may provide a theoretical basis for the rational design of more effective self-assembly vector free nano drugs and the feasibility of clinical application.