Abstract: Two-dimensional (2D) nanochannel membranes via the ordered self-assembly of 2D nanosheets offer great opportunities for developing efficient and robust devices for osmotic energy harvesting due to their controllable channel height and high density of orderly oriented channel. However, owing to the limited polar group density of nanosheets, conventional 2D nanochannel membranes can not effectively concentrate counterions, which leads to the low concentration of charge carriers and thus the low value of ion flux. Herein, graphene oxide (GO) and poly(sodium 4-styrenesulfonate) (PSS) were orderly stacked into GO-PSS composite nanochannel membranes, driven by the directional field of fluid flow. The resulting membranes feature 2D channels intercalated with abundant surface charges. The effect of ionic strength, salt concentration gradient, PSS content, and polyelectrolyte type on the transmembrane ionic transportation and osmotic energy harvesting of GO-PSS composite membranes was investigated. In contrast to pristine GO membranes, the incorporation of PSS simultaneously improves the ionic permeability and ion selectivity of GO-PSS composite membranes, thus leading to its higher output power density than that of pristine GO membranes. When the PSS content is 65%, the GO-PSS composite membranes offer an output power density up to 11.27 W·m⁻² by mixing seawater and river water, much higher than 3.37 W·m⁻² of conventional GO membranes. This work highlights the significance of charge density and presents a general strategy for effectively improving ion transport through two-dimensional nanochannel membranes for high-output osmotic energy harvesting.