We have previously demonstrated that dark materials (DM) in acidified molasses are effectively adsorbed to Amberlite XAD7HP resin and are eluted from the resin with 0.1 M sodium hydroxide. In this paper, we have characterized the self-assembly behavior of molasses DM by using dynamic and static light scattering in combination with isoelectric focusing and infrared absorption spectroscopy in order to better understand the resin adsorption mechanism. One of DM derivatives, X-G2, contained carboxyl and hydroxyl groups and had a weight-average molar mass of 9.39 × 10(3) to 4.42 × 10(4) at pH 2.1-11.5. The aggregates retained their spherical shape over the full pH range and the large gyration radius (66.4-80.0 nm) indicated that the inner structure was loosely packed. Furthermore, X-G2 had an isoelectric point of 1.8, and its density increased sharply at pH 5.9 and then approached a nearly constant value under alkaline conditions. In summary, the self-assembly processes of DM are controlled by intermolecular hydrogen-bonding and hydrophobic interactions. The aggregates adsorb to the resin through hydrophobic interactions and are eluted when excess carboxylate anions are generated.