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Microspheres bearing large pores are useful in the capture and separation of biomolecules. However, pore size is typically poorly controlled, leading to disordered porous structures with limited performances. Herein, ordered porous spheres with a layer of cations on the internal surface of the nanopores are facilely fabricated in a single step for effective loading of DNA bearing negative charges. Triblock bottlebrush copolymers (BBCPs), (polynorbornene‐g‐polystyrene)‐b‐(polynorbornene‐g‐polyethylene oxide)‐b‐(polynorbornene‐g‐bromoethane) (PNPS‐b‐PNPEO‐b‐PNBr), are designed and synthesized for fabrication of the positively charged porous spheres through self‐assembly and in situ quaternization during an organized spontaneous emulsification (OSE) process. Pore diameter as well as charge density increase with the increase of PNBr content, resulting in a significant increase of loading density from 4.79 to 22.5 ng µg−1 within the spheres. This work provides a general strategy for efficient loading and encapsulation of DNA, which may be extended to a variety of different areas for different real applications. [ABSTRACT FROM AUTHOR]