A one-step in situ method, termed microfluidic diffusion-induced self-assembly, for the synthesis of monodisperse ordered mesoporous silica microspheres, is reported. The method combines microfluidic generation of uniform droplets and subsequent in situ rapid solvent diffusion-induced self-assembly within the microfluidic channel. The mesoporous silica microspheres prepared in this way reveal well-ordered 2D hexagonal mesostructures with unprecedented corrugated surface morphology of disordered mesopores that are larger than 15 nm. It is speculated that the formation of an interfacial subphase and rapid diffusion of solvent to oil are attributed to the formation of the unique surface morphology. It is also shown that the surface morphology and the particle size of the mesoporous silica microspheres can be systematically controlled by adjusting fluidic conditions.