5-Fluorouracil (5-FU) is an antimetabolic of the pyrimidine derivatives that is used in chemotherapy for the treatment of several types of cancer. 5-FU have poor oral absorption and short biological half-time and strong side effects. Microneedle introduced to find a solution of problems. Microneedle device with roll was manufactured for transdermal delivery of various drugs. 5-FU was mixed in non-ionic surfactant such as tween 20 and tween 80. Camscope was used to analysis the permeation magnitude of treated skin by microneedle and trypan blue staining. The 5-FU solution with surfactant measured by ZETA-potential analysis system for stability of solution. The skin permeation rate of 5-FU determined by HPLC. We confirmed that cross treated skin was dyed more deeply than parallel treated skin through trypan blue staining. The results indicate that skin permeation rate of 5-FU was increased with the treatment types and treatment times.