The paeonol proniosomes ointment and ordinary ointment were administered to rats. Physiological saline served as perfused solution. The perfusion rate was 5 mL x L(-1) and the microdialysis samples were collected every 20 min intervals. The paeonol concentration in perfused solution was determined by HPLC. Investigation of the pharmacokinetics of paeonol proniosomes ointment and ordinary ointment by the skin-blood synchronous microdialysis coupled with HPLC is reported in this study. The results show that the recovery was (54.80 +/- 1.50)% in vitro and (54.58 +/- 4.61)% in vivo. The results showed that paeonol proniosomes ointment significantly raised the drug concentrations in skin more than the paeonol ordinary ointment. The paeono proniosomes ointment has less drugs into the blood as the ordinary ointments in blood, but its blood drug concentrations were steadier. The paeonol proniosomes ointment may be developed into a new preparation.