The present work investigates the development of water/mixed nonionic surfactant/co-surfactant/cyclohexane reverse microemulsions (RM) suitable for nanoparticles synthesis. The mixture of Span 80 (oil soluble) and Tween 80 (water soluble) was selected as the surfactants. Optimum formulation of RM was obtained by using the Box-Behnken (3(3)) experimental design method to evaluate the effect of three independent process variables, i.e., pH, Span 80 wt% in surfactant mixture, and propyl alcohol wt% in mixture of cyclohexane and propyl alcohol, on the preferred responses: average droplet size (ADS) and polydispersity index (PDI) of droplets. The model was validated experimentally based on an ANOVA table, and was optimized to reach the optimum formulation to yield the ADS and PDI for RMs. The determination coefficient (R-2) values of 0.991 for ADS and 0.975 for PDI show that Box-Behnken design is a useful platform for the optimization of RMs formulation. Finally, iron oxide nanoparticles were synthesized under the optimum RM conditions and the uniform nanoparticle distribution with an average particle size of 2.1 +/- 0.49 nanometer and a polydispersity of 0.06 +/- 0.011 were obtained. (C) 2017 Published by Elsevier B.V. on behalf of Institution of Chemical Engineers.