It was found for the first time that the existence of hindrance units of sodium p-styrenesulfonate (SSS) on the polymer backbone could increase the hydrophobic content of the polymers on the premise of water solubility. A novel series of brush-like amphiphilic statistical tripolymers were designed and prepared by polymerization of amphiphilic macromonomer 2-(acrylamido)-dodecane sulfonic acid (AMC(12)S, 10 to 90 mole fraction), with SSS (0 to 5 mole fraction), and 2-(acrylamido)-2-methylpro-panesulfonic acid (AMPS). The self-assembly behaviors of these tripolymers were investigated using a combination of steady-state fluorescence, dynamic light scattering (DLS), and transmission electron micrograph (TEM) methods, mainly focusing on the effect of varying unit contents along the polymer backbone on the associative performance. The increase of amphiphilic units results in a simultaneous decrease of the microdomain polarity and the assembly size. Furthermore, the increase of hindrance units leads to an increase of assembly size but no change of microdomain polarity, manifesting a further aggregation of the polymer assemblies. The further aggregation partly results from the stretch facilitation of polymer chains by the hindrance units, and the facilitation effect works more intensely for the polymers with less amphiphilic units.