For the simultaneous delivery of antisense oligonucleotides and their effector enzymes into cells, nanosized vesicular polyion complexes (PICs) were fabricated from oppositely charged polyion-pairs of oligonucleotides and poly(ethylene glycol) (PEG)-b-polypeptides. First, the polyion component structures were carefully designed to facilitate multimolecular (or secondary) association of unit PICs for non-covalent (or chemical crosslinking-free) stabilization of vesicular PICs. Chemically modified, single-stranded oligonucleotides (SSOs) dramatically stabilized the multimolecular associates under physiological conditions, compared to control SSOs without chemical modifications and duplex oligonucleotides. In addition, a high degree of guanidino groups in the polypeptide segment was also crucial for the high stability of multimolecular associates. Dynamic light scattering and transmission electron microscopy revealed the stabilized multimolecular associates to have a 100 nm-sized vesicular architecture with a narrow size distribution. The loading number of SSOs per nanovesicle was determined to be ~2,500 by using fluorescence correlation spectroscopic analyses with fluorescently labeled SSOs. Furthermore, the nanovesicle stably encapsulated ribonuclease H (RNase H) as an effector enzyme at ~10 per nanovesicle through simple vortex-mixing with preformed nanovesicles. Ultimately, the RNase H-encapsulated nanovesicle efficiently delivered SSOs with RNase H into cultured cancer cells, thereby eliciting the significantly higher gene knockdown compared with empty nanovesicles (without RNase H) or a mixture of nanovesicles with RNase H without encapsulation. These results demonstrate the great potential of non-covalently stabilized nanovesicles for the codelivery of two varying biomacromolecule payloads for ensuring their cooperative biological activity.