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Coagulation-based harvesting has been widely used in microalgal biomass harvesting. However, the coagulant contamination in the harvested biomass may negatively affect the applications in feedstock processing for food, feed, and fuel. In this study, extracellular polymeric substances (EPSs) were derived from microalgae, Scenedesmus acuminatus, and then used as a bioflocculant to aid the flocculation of the same algae. The results show that the alum coagulant (Al3+) usage was significantly reduced from 77.6 to 4.5 mg g–1 when adding this EPS bioflocculant at a dose of 3.2 mg g–1, which potentially reduces the chemical cost from $282 per metric ton to $71 per metric ton dry biomass that is harvested. To analyze the compositions of this bioflocculant, molecular fractionation was performed. The functional fractions such as protein-like and humic-like organic substances were characterized by fluorescence excitation–emission, followed by polysaccharide analysis. Low-MW (<3 kDa) EPS contributed to the flocculation process more than the large-MW fractions. Low-MW EPS contained higher contents of glucose and mannose in the polysaccharide that influence the interactions of the algae and the alum coagulant. Microalgal-derived bioflocculants may open up new avenue toward the low-cost and sustainable bioflocculation processes for algal and other biomass separation.

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