Microalgae as the third generation of biomass sources for biofuel production has a promising future, but its economic challenges have not been resolved yet. One of the major challenges in order to practical implementation of biofuel production is separation of microalgae from culture medium, since this step devotes a significant part of the energy and time consumption. Although various methods have been reported for harvesting, electro-coagulation-flotation (EC) method was preferred due to the simplicity and capability of process scale up. In this study, the effect of three parameters, i.e., initial pH, electric current density and duration on microalgae separation efficiency and operating costs, as the response variables, was investigated by applying "Response Surface Methodology" (RSM) technique. It was revealed that duration was the most effective parameter on the microalgae separation efficiency, as well as the operational costs, wherein high efficiency was resulted in long duration. On the other hand, the high amount of electric current density caused to high coagulation, flotation rates, and also high power consumption. Although the pH of solution had a little impact on operating cost compared to the duration and current density, the natural condition, pH = 6, was recommended. In optimal point; current density = 1.6 mA/cm(2) for 17.65 mm, deprived of demanding to adjust pH, the microalgae separation efficiency was obtained 96.8%. Since the EC is a substitute for CC (chemical coagulation), its performance was evaluated on microalgae separation efficiency. The results demonstrated that the optimal coagulant concentration and pH was 450 mg/l and 8 respectively. At the optimal conditions, microalgae separation efficiency was resulted in 85%. Consequently, due to less sensitivity to pH changes, more flexibility and lower cost, the EC was introduced as a more efficient technology than CC Technology. All of these benefits recommend this method as the promising technique of microalgae harvesting in large-scale. (C) 2015 Elsevier B.V. All rights reserved.