Colloidal interactions between magnetic nanoparticles (NPs) and algal cells are of paramount significance to magnetophoretic separation of algal biomass from water. This study evaluated the size effect of magnetic NPs (MNPs) coated with polyethylenimine (PEI) on the separation efficiency of Scenedesmus dimorphus as well as on the recovery efficiency of MNPs from algal biomass. Results showed that algal harvesting efficiency (HE) increased from ca. 60% to 85% as the diameter of PEI-coated MNPs increased from 9 to 53 nm. Likewise, algal recovery capacity (algae/MNPs, w/w) also showed the same size dependence. But a large size (247 nm) led to a decline of algal HE, which was correctly interpreted by a settling model that predicts large sizes of MNPs could eventually reduce the settling velocity under magnetophoresis. The extended Derjaguin-Landau-Verwey-Overbeek theory revealed that the particle size and PEI coating both influenced the interaction energies (e.g., energy barrier) between MNPs and algae. Particularly, PEI coating significantly reduced the energy barrier between MNPs and algae and thereby increased their heteroaggregation and algal HE. Moreover, PEI-coated MNPs were recovered from the harvested algae biomass through a chemical-free ultrasonic method, and the recovery efficiency appeared to be higher for larger MNPs. Overall, the synthesized sizes of applied MNPs will not only affect algal HE but also have economic implications on magnetophoretic algal separation technologies.