Osmotic energy, existing between sea water and river water, is a clean, renewable, and sustainable energy source that can be converted into electricity using ion-exchange membranes (IEMs) based on reverse electrodialysis. Asymmetric IEMs with thin selective layers have great potential in the osmotic energy conversion. However, there is still a tradeoff between ion selectivity and ion conductivity in the selective layer. Here, we demonstrate an up-scalable asymmetric nanochannel membrane with a crosslinked monomolecular selective layer that enhances osmotic energy conversion. A monomolecular layer of hyperbranched polyethyleneimine (h-PEI) is precisely grafted at the end of carboxylic nanochannels. The grafted h-PEI layer is fully expanded in the real aqueous condition, leading to unexpected permselectivity decrease. To avoid this, the h-PEI layer is in situ crosslinked with cyanuric chloride (CC), resulting in a dramatic decrease in thickness. The resultant asymmetric nanochannels with a crosslinked monomolecular h-PEI layer show increased Cl − permselectivity and conductivity, achieving enhanced osmotic energy conversion with a power density of 11.9 W/m 2 at a 500-fold salinity gradient, nearly twice as high as the pre-crosslinked membrane. The membrane design concept would pave the way for osmotic energy conversion and could be extended to other separation membranes.