The synthesis of mesoporous nanoparticles with controllable structure and organic groups is important for their applications. In this work, yolk–shell-structured periodic mesoporous organosilica (PMO) nanoparticles simultaneously incorporated with ethane-, thioether-, and benzene-bridged moieties are successfully synthesized. The preparation of the triple-hybridized PMOs is via a cetyltrimethylammonium bromide-directed sol–gel process using mixed bridged silsesquioxanes as precursors and a following hydrothermal treatment. The yolk–shell-structured triple-hybridized PMO nanoparticles have large surface area (320 m2 g–1), ordered mesochannels (2.5 nm), large pore volume (0.59 cm3 g–1), uniform and controllable diameter (88–380 nm), core size (22–110 nm), and shell thickness (13–45 nm). In vitro cytotoxicity, hemolysis assay, and histological studies demonstrate that the yolk–shell-structured triple-hybridized PMO nanoparticles have excellent biocompatibility. Moreover, the organic groups in the triple-hybridized PMOs endow them with an ability for covalent connection of near-infrared fluorescence dyes, a high hydrophobic drug loading capacity, and a glutathione-responsive drug release property, which make them promising candidates for applications in bioimaging and drug delivery.