In this work, a novel flocculation process by using nano-Fe3O4 coated with polyethyleneimine (Fe3O4@PEI) as magnetic seeds was developed to harvest the microalgae cultivated in urban sewage. Experiment results indicated that the harvest efficiency of Chlorella pyrenoidosa (0.5 g/L) was 98.92 ± 0.41% under the optimal conditions of Fe3O4@PEI dose of 20 mL/L, flocculation time of 20 min, and stirring speed of 800 rpm (3 min), while that of Scenedesmus obliquus (0.4 g/L) was 98.45 ± 0.35% under a Fe3O4@PEI dose of 16 mL/L, flocculation time of 15 min, and stirring speed of 730 rpm (3 min). Moreover, the process did not reduce the lipid content of microalgae and quality of biodiesel. After microalgae harvest, Fe3O4@PEI could be recovered by ultrasonication, re-wrapped with polyethyleneimine and reused to reduce operational cost.