The equilibrium potentiometric (EMF) response of a previously reported polymer membrane-based heparin sensitive electrode toward heparin and other macromolecular polyanions is examined in detail. The precise structure of the lipophilic tetraalkylammonium anion-exchange sites doped within the plasticized poly(vinyl chloride) membrane phase of the sensor is found to play a critical role in the extraction of the polyanions into such membranes. The influence of polyion charge density and molecular weight (chain length) for a series of structurally similar polyanions (sulfonated/carboxylated polysaccharides) on the membrane’s EMF response is also studied. It is found that the membrane potential becomes more negative (for given mass/volume concentration of polyion) with an increase of polyion molecular weight up to 3800 for heparin-like structures. In addition, a greater overall equilibrium response is observed as the charge density of the polyanion increases. The observed role of polyanion structure on the membrane’s equilibrium EMF response can be explained by a favorable ion-pair formation between the Lipophilic anion-exchanger sites in the membrane and the extracted polyanions. The free energy change for such ion-pair formation can be estimated by comparing the membrane’s equilibrium EMF response toward a given polyion to that of a second polymeric membrane doped with anion-exchange sites that do not form strong ion pairs with polyanionic macromolecules. The influence of the sample background electrolyte concentration on the extraction behavior of different polyions is also examined.