A novel micelle/precursor co-templating assembly strategy is successfully developed to synthesize large-pore ultrasmall mesoporous organosilica nanoparticles (MONs). Furthermore, elaborately designed MONs with a cell-penetrating peptide (TAT) (MONs-PTAT) are constructed for highly efficient intranuclear gene delivery. They exhibit a high loading capacity, improved protection for the loaded gene, and enhanced transfection efficiencies of EGFP plasmid (pEGFP).