This study examined a novel ethoxy-segment-regulated hydrophobic associative amphiphilic copolymer, P(AA-AAEO_n), and systematically evaluated its solution self-assembly behavior and enhanced oil recovery (EOR) performance. The influence of ethylene oxide (EO) chain length and polymer concentration on particle size distribution and aggregation morphology was analyzed using dynamic light scattering (DLS). The results revealed a concentration-dependent transition from intramolecular to intermolecular association, accompanied by a characteristic decrease followed by an increase in hydrodynamic diameter. At a fixed AA:AAEO_n molar ratio (400:1), increasing EO segment length increased aggregate size and improved colloidal stability. Viscometric analysis showed that longer EO chains markedly increased molecular chain flexibility and solution viscosity. Interfacial tension measurements demonstrated superior interfacial activity of P(AA-AAEO_n) compared to polyacrylic acid (PAA), and longer EO chains further reduced oil–water interfacial tension. Emulsification tests verified its strong ability to emulsify crude oil. Sandpack flooding experiments and micromodel studies demonstrated effective conformance control and high displacement efficiency, achieving up to 30.65% incremental oil recovery. These findings offered essential insights for designing hydrophobic associative polymers with tunable interfacial properties for EOR applications.