This study is to prepare the microemulsion-based gel based on the W/O microemulsion and fluorouracil (5-Fu) as a model drug to study the transdermal characterization and observe its skin irritation of the microemulsion-based gel in vitro.  IPM acted as oil phase, AOT as surfactant, Tween 85 as cosurfactant, water was added dropwise to the oil phase to prepare W/O microemulsion at room temperature using magnetic stirring, then 5-Fu powder was added.  The gelatin was used as substrate to prepare 5-Fu microemulsion-based gel.  The permeation flux of 5-Fu from 5-Fu microemulsion-based gel across excised mice skin was determined  in vitro using Franz diffusion cell to study the influence of the amount of gelatin and the drug loading capacity.  Refer to 5-Fu cream, the irritation of microemulsion and microemulsion-based gel on the rat skin was studied.  Based on the water/AOT/Tween 85/IPM microemulsion, only the gelatin can form the microemulsion-based gel.  At 25 ℃, 32 ℃ and 40 ℃, the amount of gelatin required for the formation of microemulsion-based gel were 7%, 14% and more than 17%, respectively.  The 12 h transdermal cumulated permeation amount of 5-Fu from  microemulsion-based gel containing 14% gelatin and 0.5% drug loading were (876.5 ± 29.1) μg·cm⁻², 12.3 folds and 4.5 folds more than 0.5% 5-Fu aqueous solution and 2.5% (w/w) 5-Fu cream, respectively.  Microemulsion- based gel exhibited some irritation, but could be subsided after drug withdrawal.  Microemulsion-based gel may be a promising vehicle for transdermal delivery of 5-Fu and other hydrophilic drug.