Two-dimensional (2D) nanomaterials have been the focus of substantial research interest recently owing to their fascinating and excellent properties. However, 2D porous materials have remained quite rare due to the difficulty of creating pores in 2D nanostructures. Here, we have synthesized a novel type of single-layered 2D mesoporous TiO2 nanosheets with very uniform size and thickness as well as ordered mesostructure from an unprecedented hydrothermal-induced solvent-confined assembly approach. The F127/TiO2 spherical monomicelles are first formed and redispersed in ethanol and glycerol, followed by a hydrothermal treatment to assemble these subunits into single-layered 2D mesostructure owing to the confinement effect of highly adhered glycerol solvent. The obtained 2D mesoporous TiO2 nanosheets have a relative mean size at around 500 × 500 nm and can be randomly stacked into a bulk. The TiO2 nanosheets possess only one layer of ordered mesopores with a pore size of 4.0 nm, a very high surface area of 210 m2 g-1 and a uniform thickness of 5.5 nm. The thickness can be further manipulated from 5.5 to 27.6 nm via simply tuning precursor concentration or solvent ratio. Due to the well-defined 2D morphology and large mesoporosity as well as crystalline anatase mesopore walls, these uniform TiO2 nanosheets are capable of providing large accessible voids for sodium ion adsorption and intercalation as well as preventing volume expansion. As expected, these mesoporous TiO2 nanosheets have exhibited an excellent reversible capacity of 220 mAh g-1 at 100 mA g-1 as sodium-ion battery anodes, and they can retain at 199 mAh g-1 after numerous cycles at different current densities. The capacity is retained at 44 mAh g-1 even at a large current density of 10 A g-1 after 10 000 cycles, demonstrating a remarkable performance for energy storage.