Abstract: Macromolecular drugs have attracted considerable attention owing to their high specificity, multifunctionality, and favorable biocompatibility. However, their clinical translation is severely hampered by inherent limitations, including poor stability and insufficient membrane-crossing ability. Although nanocarriers can improve the bioavailability and targeting of these drugs, achieving efficient intracellular delivery with controlled release remains a critical challenge. In this study, we design and synthesize amphiphilic poly(amino acid) derivatives with distinct conformations. These polymers self-assemble into polymersomes that efficiently encapsulate hydrophilic macromolecular drugs. Compared to their β-sheet counterparts, α-helical poly(amino acid) polymersomes exhibit relatively weaker intermolecular interactions, resulting in superior membrane permeability and thus more effective release of the encapsulated cargo. Remarkably, the α-helical conformation also imparts enhanced membrane activity, which not only facilitates efficient uptake by tumor cells but also promotes endo-lysosomal escape, leading to improved intracellular release and distribution of macromolecular drugs. This study proposes a novel strategy for macromolecular drug delivery, and the engineered α-helical poly(amino acid) polymersomes emerge as a promising category of nanocarriers with extensive translational application prospects.