High-throughput synthesis of well-defined polymer nano-objects has long been an attractive yet challenging topic in the area of polymer chemistry and material science. Herein, we report an enzyme-assisted photoinitiated polymerization induced self-assembly (photo-PISA) approach to prepare well-defined AB diblock or ABC triblock copolymer nano-objects at room temperature in open vessels and multiwell plates. Kinetic studies indicated that the presence of glucose oxidase (GOx) endowed the polymerizations with excellent oxygen tolerance. Good control was maintained during: the enzyme-assisted photo PISA process. This method facilitates high-throughput PISA, allowing for the construction of a detailed phase diagram in a rather short time. We also demonstrate the potential bio-related application of this method by the successful encapsulation of horseradish peroxidase (HRP) and bovine serum albumin (BSA) into self-assembled polymer vesicles without compromising protein activities. This robust oxygen-tolerant PISA approach leads to unprecedented access to well-defined polymer nano objects for nonexperts.