Energy harvesting based on salinity gradients between oceans and rivers can provide a clean, stable, and continuous electric output. However, it is highly dependent on the excellent performance of biomimetic ion-selective membranes. The graphene oxide (GO) membrane, as a 2D material-based layered-structure membrane, offers the possibility of efficient salinity gradient energy conversion. However, unstable stratification in aqueous solutions and the limited number of charged functional groups make practical applications difficult. A conventional symmetric structure will also reduce the effective salinity gradient owing to ion enrichment on the low-concentration side. In this study, we pre-modified GO to create an asymmetric ion-selective membrane with opposing charges. Hyperbranched polyethyleneimine (PEI) and polyacrylic acid (PAA) as surface charge donors and crosslinkers stabilise the interlayer spacing of GO and increase charge density, achieving up to 3.4 W/m2 power density under sea water and river water. Furthermore, theoretical calculations show that membranes with asymmetric structure and opposite charges can eliminate the effect of ion enrichment on the low-concentration side, thereby avoiding power consumption. This system contributes to a further step toward the application of salinity energy conversion.