<p>Nanofactories are artificial vesicles that create protected, microscopic reaction environments for encapsulated enzymes. The nanostructure of the vesicles shields the internal enzymes while allowing small substrates to permeate the membranes for catalytic reactions. A key challenge in the synthesis of nanofactories is achieving efficient enzyme encapsulation. Here, we constructed self-assembled vesicles from a thermoresponsive peptoid-based block copolymer, which achieved a high enzyme encapsulation efficiency of more than 50%. In contrast, conventional methods typically yield efficiencies of only a few percent. We hypothesized that this high performance stems from a temperature-induced coacervate-to-vesicle phase transition, where enzymes are first partitioned into a polymer-rich coacervate phase before being entrapped within vesicles upon heating. The resulting enzyme-loaded vesicles acted as robust nanoreactors, shielding enzymes from external proteases, while exhibiting selective permeability that enabled substrate sorting on the basis of physicochemical properties. This highly efficient encapsulation strategy, leveraging the phase transition of biocompatible materials, resolves a long-standing bottleneck in nanoreactor development. Such advanced nanoreactors could pave the way for novel biomedical applications, including in vivo therapeutic synthesis and the construction of sophisticated artificial organelles.</p>