Transfersomes is one of the most effective nanotechnology-based formulation for transdermal delivery of minoxidil (MXD), however the potential use was limited by its low drug content (DC) and entrapment efficiency (EE). In this study, efforts have been made to improve DC and EE of MXD in transfersomes for effective topical treatment of alopecia. By dissolving both MXD and edge activator (Tween 80) in aqueous phase than either or both of them co-dissolving in organic solvent with lipids (phospholipid and cholesterol with mass ratio of 5:1), the DC and EE of MXD transfersomes prepared by thin film hydration method were significantly increased. The optimized transfersomes (MT) and its gel (MT_gel) with mass ratio of drug, Tween 80 and lipids 3:1:18 have a spherical morphology without aggregation, and a particle size of not more than 100 nm. The differential scanning calorimetry measurements revealed that the drug in MT was in the non-crystalline state. MT and MT_gel exhibited better in vitro transdermal penetration ability and hair of C57BL/6 mice growth-promoting capacity than commercial formulations. MT_gel has non-irritation and is better than MT in skin adhesion, stability, potential for skin storage and hair growth promotion ability, which could endow it to be a promising strategy for topical treatment of alopecia.