As part of our continuing research work, studies toward the self-aggregation behaviour of amphiphilic triblock copolymers containing β-CD-Pluronic F127 polyrotaxane as a central block and hydrophilic brush-like PPEGMA as flanking blocks in aqueous solution were conducted by using dynamic and static light scattering (DLS and SLS) analyses and transmission electron microscopy (TEM). These self-aggregates are characterized by a unique random coil structure. Their hydrodynamic radius (R_h) and radius of gyration (R_g) decrease as the number of the entrapped β-CD molecules increases, while the core of the random coils becomes looser due to the increase of the rigidity and steric hindrance of the brush-like polymer chains. Interestingly, it was observed that the morphology of the aggregates changes greatly after loading amphotericin B (AmB). According to the DLS/SLS and TEM results, it was speculated that a solid sphere is formed, and that the density of spheres increases as the number of entrapped β-CDs increases. For these self-aggregates, as the number of entrapped β-CDs increases, their drug-loading content (DLC) and drug-loading efficiency (DLE) for AmB increases, while their hemolytic activity against rabbit erythrocytes decreases. It appears that the multiple hydrogen-bonding interactions between AmB and the entrapped β-CDs make a significant contribution to the morphology change of the self-aggregates and their high loading capability for AmB.