Block copolymer vesicles loaded with active compounds have been employed as decent candidates to mimic complex biological systems that attract considerable interest in different research communities. We herein report a visible light-initiated seeded reversible addition-fragmentation chain transfer (RAFT)-mediated polymerization-induced self-assembly (PISA) for in situ preparation of enzyme-loaded cross-linked block copolymer vesicles without compromising the bioactivity. Permeability of the vesicular membrane can be regulated through changing the solution temperature, allowing further control over the enzymatic reaction rate of enzyme-loaded vesicles. Finally, non-cross-linked thermoresponsive block copolymer vesicles that can transform into worm-like micelles at low temperature are also prepared by this method, allowing the release of bimacromolecules from the vesicles under mild conditions.