For commercial scale algal biorefining, harvesting cost is a major bottleneck. Thus, a cost-effective, less-energy intensive, and efficient harvesting method is being investigated. Among several harvesting methods, magnetic flocculation offers the benefits of modest operation, energy savings and quick separation. This study aims to develop novel magnetite-(FeO) nanoparticles (MNPs) of 20 nm average size and their high reusability potential to reduce the harvesting cost of microalgae biomass. The MNPs were synthesized and characterized using FTIR, Zeta analyzer, and SEM before performing on Chlorella sorokiniana Kh12 and Tu5. For maximum harvesting efficiency >99%, the optimal culture pH, MNPs concentration, and agitation speed were 3, 200 mg/L, and 450 rpm, respectively. Subsequently, MNPs were recovered via alkaline treatment and reused up to 5 cycles as they retained their reactivity and harvesting efficiency. The studied MNPs-based harvesting method could be adopted at a commercial scale for cost-effective algae biorefinery in the future.