Abstract Background Alopecia has become a very common disease that many people around the world are suffered. Minoxidil (MXD) is the most well-known commercialized drug in its treatment. However, in the case of MXD administration, there are some problems with low efficiency of transdermal delivery and additional side effects. Method MXD and Rhodamine B (Rho B) are encapsulated in poly(Lactide-co-Glycolide) grafted hyaluronate nanoparticles (HA-PLGA/MXD NPs, HA-PLGA/Rho B NPs) which is prepared with W/O/W solvent evaporation method. After then, the investigation is carried out to confirm the feasibility of NPs in alopecia treatment. Results Both of HA-PLGA/MXD NPs and HA-PLGA/Rho B NPs are successfully prepared. In addition, it is confirmed that HA-PLGA NPs sufficiently delivered to cells without any significant cytotoxicity by cell viability, cellular uptake and skin permeation test. Conclusion Taken together, HA-PLGA NPs as a transdermal delivery carrier to hair follicle cells can be exploited to develop the efficient and effective platform of transdermal drug delivery for the treatment of various diseases.