Abstract: As a non-invasive drug delivery route, transdermal drug delivery system (TDDS) has several advantages over conventional drug delivery methods, such as better patient compliance and no first-pass effect. The application of nanotechnology has further expanded the range of drugs choices in TDDS and improves the therapeutic effects of drugs, forming a valuable and attractive way of drug delivery. At present, the nano-formulations applied in TDDS include nanoemulsions, lipid-based nano-vesicles, lipid nanoparticles, polymer nanoparticles, nanocrystals, lyotropic liquid crystal nanoparticles, and so on. In this article, we introduced skin barriers and common means of penetration enhancement in transdermal delivery. We also reviewed the research progress of the nano-formulations applied in TDDS and their combined applications with physical methods for penetration enhancement, aiming to provide a reference for the further study of nano-formulations in transdermal delivery.