Background: An intricate autoimmune disease called alopecia areata causes hair loss. Even though there are numerous therapeutic alternatives, the most of them have significant side effects and/or poor efficacy. This promotes the continuous development of novel therapies. The purpose of this study was to compare intralesional injection of 5-fluorouracil with transdermal delivery of the drug using fractional CO2 laser for the treatment of alopecia areata. Methods: The study was clinical trial. It included 52 AA patients were divided into two groups each group 26 patients Group I (Transdermal of 5-flurouracil following fractional laser) and Group II (Cases injected with intralesional 5-Flurouracil). Clinical examination was done with assessment of disease severity at baseline and each visit using Severity of Alopecia Tool Score (SALT). Digital photographs of the lesions were performed at baseline and at weeks 2,4,6,8 &12 for results evaluation. Results: Regarding response to treatment : There was a statistical significant difference among both groups regarding to response to treatment, as 15.4% of group II showed no response versus 7.7% of group I, while 61.5% of group I showed excellent response versus 23.1% of group II. And there was a statistical significance decrease in SALT score in both groups post treatment compared to pre by (65.5% & 34.1% respectively). Conclusions: Treatment of Alopecia Areata using transdermal delivery of 5-Fluorouracil by fractional CO2 laser showed excellent response compared by intralesional injection of 5-Fluorouracil