Objective To explore the effects of prescription optimization of 5-fluorouracil (5-FU) patch and electrostatic field of the negative polarity electret on the in vitro release behavior of 5-FU from the 5-FU patch. Methods The corona charging technology at ambient temperature was applied for preparation of －1 000 V and －2 000 V negative polarity electrets. The negative polarity electret 5-FU patch was prepared by using Eudragit E100 as pressure-sensitive adhesive (PSA), tributyl citrate as plasticizer, and water-soluble azone as chemical permeation enhancer. We used the single factor method to investigate the influences of PSA, plasticizer, water-soluble azone and negative polarity electret on the relevant parameters of the 5-FU patch. The optimal prescription was screened by orthogonal design test. We also studied the influence of negative polarity electret on the in vitro release behavior of 5-FU from the 5-FU patch by using modified Franz diffusion cell and high-performance liquid chromatography. Results (1) The optimal prescription was fitting when the mass of Eudragit E100 was 0.25 g, the mass ratio of plasticizer to Eudragit E100 was 55:100 and the mass fraction of water-soluble azone was 3%. (2) Both the －1 000 V and －2 000 V negative polarity electrets used in this study promoted the in vitro release of 5-FU from the 5-FU patch; and the promoting effect was proportional to the surface potential of the electret. (3) Compared with the single use of 3% water-soluble azone, negative polarity electret combined with 3% water-soluble azone significantly increased the in vitro release of 5-FU from the 5-FU patch (P<0.05). Conclusion Negative polarity electret and water-soluble azone can reduce the holding capacity of the 5-FU patch and promote the in vitro release of 5-FU from the 5-FU patch, and the promoting effect is more significant in the combination of them.