Excess sludge produced by wastewater treatment plants contains extracellular polymeric substances (EPSs; 10-40 dry wt%) that mainly comprise polysaccharides and proteins and can be used as bioflocculants, adsorbents for heavy metal ions, and soil conditioners. Herein, we investigated the separation of EPS constituents using unstirred and stirred dead-end microfiltration in the presence of Ca2+, with an aqueous solution of sodium alginate (SA) and bovine serum albumin (BSA) employed as an EPS model. Acceptable separation efficiencies were obtained utilizing filter papers with micropores of various sizes, which retained calcium alginate aggregates formed due to the SA-Ca2+ interaction but allowed BSA to be filtered out. Notably, a certain minimal Ca2+ concentration was required to achieve the above separation, with the separation efficiency being independent of the SA:BSA concentration ratio at Ca2+ concentrations exceeding the threshold value. The filtration resistance decreased with increasing pore size, Ca2+ concentration, and BSA content. Rather unexpectedly, variation of filtration surface shear could not be used to improve the filtration separation efficiency and reduce fouling in spite of the revolution rate, since the disturbing flow forced the aggregates into the filter pores. The polysaccharide in the EPSs was separated effectively by microfiltration with the addition of Ca2+. Thus, the obtained results provide further insight into the microfiltration-aided separation and recovery of polysaccharides and proteins in EPSs.