A novel approach for harvesting Scenedesmus sp. microalgae from real wastewater by using adsorbents of magnetite-based nanoparticles (Fe3O4 NPs) was tested in this study for the first time for this microalgae. Using these NPs, the harvesting efficiency was even higher than 95%. The optimal conditions (0.14 gNPs/L, a short magnetic separation time of only 8 min and 27 min of contact time) were found using the response surface methodology. The best fitting of the adsorption equilibrium results was achieved by the Langmuir isotherm model, and the maximum adsorption capacity for Scenedesmus sp. reached 3.49 g dry cell weight (DCW)/g Fe3O4 NPs. Zeta potential measurements and the Dubinin-Radushkevich isotherm model analysis pointed out that the main adsorption mechanism between Scenedesmus sp. cells and Fe3O4 NPs was electrostatic interaction. Finally, Fe3O4 NPs were six times successfully reused by combining an alkaline treatment with an ultrasonication process, which implies microalgae lysis. The results herein obtained highlight the potential for magnetic separation of microalgae from wastewater, which is capable of reaching a high harvesting efficiency in a very short time.