Layered titanate nanosheets (HT-ns) are potential two-dimensional materials and have wide applications due to their unique structure and low cost. In this study, the HT-ns with different lateral sizes were successfully prepared by exfoliating HT crystals with different lateral sizes, which were prepared by the flux method and ion-exchange reaction. The HT-ns were used to fabricate inorganic nanofiltration membranes by restacking the nanosheets on a porous substrate. The effects of the lateral size of HT-ns on the solute rejection performance and nanostructure of HT-ns membranes (HT-Ms), as well as the effect of surface modification of the substrate on HT-M stability, were investigated. The negatively charged substrate surface was changed to a positively charged surface by surface modification to enhance the bonding between the substrate and HT-ns, which improved the HT-M stability. A series of large-area ultrahydrophilic HT-Ms were fabricated by a facile dry deposition method on surface-modified substrates using the HT-ns with different lateral sizes. The rejection rates for inorganic salts and organic dyes increased with the increase in the lateral size of HT-ns used in HT-M fabrication, and a rejection rate of 99.8% was achieved for rhodamine B. A separation mechanism for HT-Ms was proposed to explain the separation performance, which is also suitable for other metal oxide nanosheet nanofiltration membranes.