Objective To compare the effects of chemical penetration enhancers and negative electret in promoting percutaneous penetration of 5-fluorouracil (5-FU) through rat scar and dorsal skin in vitro, so as to lay a foundation for preparing delayed-release 5-FU electret transdermal patch. Methods The in vitro transdermal behaviors of 5-FU through rat scar and dorsal skin under the actions of 1% azone, 10% ethyl oleate, -1 000 V electret, -1 500 V electret and -2 000 V electret were studied using improved Franze diffusion cell and high performance liquid chromatography (HPLC). Results (1) Both 1% azone and 10% ethyl oleate promoted the penetration of 5-FU through rat scar and dorsal skin, with the promoting effect of 10% ethyl oleate being better than that of 1% azone. (2) Although the transdermal behaviors of 5-FU were similar through scar skin and dorsal skin at the presence of chemical enhancers, the cumulative penetration amount through the scar skin was less than that through the dorsal skin. (3) The negative electrets used in this study had satisfactory penetration promoting effect, with the promoting effect from strong to weak being -2 000 V electret > -1 500 V electret > -1 000 V electret. Moreover, the scar skin also had less cumulative penetration amount of 5-FU than that of the dorsal skin under the action of electrets. Conclusion Both the chemical enhancers and electrets can enhance the transdermal delivery of 5-FU. 10% ethyl oleate and -2 000 V electret have the best enhancing effect on 5-FU transdermal delivery and can be considered for preparation of 5-FU electret transdermal patch.