Abstract: OBJECTIVE To develop tacrolimus ointment with the same transdermal permeability as the original preparation. METHODS The content of tacrolimus was determined by LC-MS/MS. Theological parameters(shear stress, compound viscosity, and etc.) and cumulative release amount of tacrolimus ointment were used as the evaluation index to screen the formulation of tacrolimus ointment. The improved Franz diffusion cells and the skin of miniature pig were used to investigate the percutaneous permeability of tacrolimus ointment. RESULTS The self-made tacrolimus ointment and original preparation had shear stress(shear velocity:30 s^-1) of 145.8, 141.8 Pa, compound viscosity of 16.11 and 15.88 Pa·s, release velocity of (1 400±243)ng·cm^-2·h^-1/2 and (1 243±133)ng·cm^-2·h^-1/2, percutaneous penetration velocity of (40.28±3.11)ng·cm^-2·h^-1and (37.61±9.09)ng·cm^-2·h^-1, residue in skin of (9.515±1.096)ng·mg^-1 and (8.463±1.770)ng·mg^-1, respectively. CONCLUSION There is no significant difference in the rheology, release velocity, and transdermal permeability between the self-made tacrolimus ointment and the original preparation.