Uniform mesoporous TiO2 nanospheres were successfully developed via an interfacial confined formation process for application in dye-sensitized solar cells. The mesoporous spherical structures greatly promote the dye-loading capacity, electron transfer, and light scattering, resulting in remarkable enhancement of the cell performance. The designed interfacial platform caused a reaction-limited aggregation of the TiO2 nanocrystals, resulting in the formation of mesoporous spherical nanostructures with sphere diameter of 216 nm and pore size of 8 nm. The oriented attachment of adjacent TiO2 nanocrystals facilitated the electron transfer process when the mesoporous TiO2 nanospheres were used as electrode films. The dye coverage was enhanced remarkably in the mesoporous spherical TiO2 samples. Owing to the enhanced light-harvesting efficiency, solar conversion efficiency was enhanced about 30% for the dye-sensitized solar cell (DSSC) based on mesoporous spherical TiO2 in comparison with that made by commercial TiO2 nanoparticles.