To further increase the drug loading (DL) of minoxidil (MXD) in transfersomes and fully utilize its role in the treatment of androgen alopecia (AGA), a strategy of drug in-cyclodextrin (CD)-in transfersomes was adopted and investigated. The sulfobutyl-substituted β-CDs (SBE-β-CD) with highest optimal binding energy was selected as the most suitable CD for MXD through molecular docking, which had high solubilization effect on MXD verified by phase solubility study. The transfersomes bearing inclusion complexes of MXD/SBE-β-CD was prepared by thin film hydration method, and the MXD/SBE-β-CD transfersomes (MXD/SCD@TFs) with the mass ratio of 10: 3: 2.5: 6 for phosphatidylcholine, cholesterol, Tween80 and MXD was selected as the optimal prescription through single factor screening, which had particle size of less than 100 nm, zeta potential of (-36.4 ± 0.11) mV. The DL of MXD/SCD@TFs was (44.02 ± 0.16)% (n = 3), which was increased nearly 3 times of MXD@TFs, resulting higher stability, in vitro skin permeation activity and drug skin retention. Importantly, the length of newborn hair and the length and number of hair follicles after 21 days of administration of MXD/SCD@TFs on AGA model C57BL/6 mice were significantly higher than those of MXD@TFs and commercial tincture (p<0.01). Obviously, improving DL is an effective means to improve the therapeutic effect of MXD transfersoms on AGA, the screening of suitable CD for MXD and the loading of inclusion complex in transfersomes provide promising strategy to achieve it.