The ability to couple self-assembly to the synthesis of materials in situ is an attractive concept for both basic and applied research. This process has been demonstrated for dense solid building blocks; however, it remains underdeveloped for porous materials. Herein, we report the one-step synthesis of mesoporous silica microspheres by self-assembling mesoporus silica nanoparticles. The nanoparticle size can be modified to control the mesopore size of microspheres, which is derived from the interstitial spacing among the nanoparticles. The assembled structures have dual hierarchy arising from both porosity and structural building blocks.