Stacking two-dimensional (2D) nanosheets into laminar membranes to create nanochannels has attracted widespread attention at fundamental and practical levels for separation technology. Constructing space-tunable and long-term stable sub-nanometer channels provide original systems for nanofluidic investigations and accurate molecular sieving. Although proof-of-concept for nanolaminate membranes has recently been demonstrated, uncontrollable swelling and the presence of pinholes prevent the scaling up of these membranes. Here, we report a scalable strategy for the preparation of non-swelling covalently functionalized molybdenum disulfide (MoS2) membranes with tunable interlayer space. The capillary height of nanochannels was precisely tuned from 3.5 to 7.7 Å, controlled by the nature of the functional groups attached on the MoS2 nanosheets, which exhibit minimal swelling in water. We evaluated the relationship between the capillary height, the surface chemistry, the stacking disorder and the sieving behaviors of the membranes in forwards osmosis (FO). We found that water permeation is strictly controlled by the capillary height of the nanochannels and the stacking disorder of the nanosheets. By combining experimental investigations and numerical simulations, we identified that the functionalization with aryl groups induces the formation of an interlayer space of 7.1 Å and interlayer stiffness as low as 5.6 eV Å-2, leading to controlled stacking defects. We report the fabrication of membranes up to 45 cm2, which demonstrate a salt rejection as high as 94.2% for a continuous operating time of 7 days. Our work presents a desalination strategy in FO with a specific energy consumption (SEC) of 4 × 10-3 kWh m-3, which compares favorably with commercial FO membranes. We anticipate that this opens avenues for the development of FO membranes for desalination based on nanolaminated structure.