Background: The mosquitoes of Culicidae family are serious vectors of several tropical diseases, such as malaria, filariasis, encephalitis, and nuisance. Control of mosquitoes and protection of people from their bites are of the most important ways to prevent transmitted diseases. Although the efficacy of N, N-diethyl-m-toluamide (DEET) is high and generally used as mosquito repellent, yet a number of biting diptera are tolerant to DEET. Furthermore, there are concerns about the safety of DEET and its allergic and toxic effects. Therefore, it is necessary to use other repellents like plant essential oils. Objectives: The aim of this work was to develop a safe repellent with a long-lasting protection based on micro-emulsion of eucalyptus essential oil. Methods: Eucalyptus globulus essential oil was obtained by water distillation in a Clevenger apparatus. The larvae of Culicidae were collected and adult mosquitoes reared for the repellency test. Preparation of micro-emulsions of Eucalyptus globulus essential oil was made by mixing the specified surfactant (Tween 80 and Span 20) and the appropriate amount of co-surfactant (propylene glycol) under the water titration method. The laboratory method, arm in cage, was used to estimate the time of protection of essential oil micro-emulsion against mosquitoes and DEET used as a standard repellent. Results: Physicochemical properties of formulated micro-emulsions were appropriate and suitable for topical application. Particle size of eucalyptus oil 15% w/w micro-emulsion was lowest. When applying eucalyptus oil micro-emulsion at concentrations of 5, 10,