Atomic thick 2D materials hold great potential as building blocks to construct highly permeable membranes, yet the permeability of laminar 2D material membranes is still limited by their irregularity sheep track‐like interlayer channels. Herein, a supramolecular‐mediated strategy to induce the regular assembly of high‐throughput 2D nanofluidic channels based on host–guest interactions is proposed. Inspired by the characteristics of motorways, supramolecular‐mediated ultrathin 2D membranes with broad and continuous regular water transport channels are successfully constructed using graphene oxide (GO) as an example. The prepared membrane achieves an ultrahigh water permeability (369.94 LMH bar−1) more than six times higher than that of the original membranes while maintaining dye rejection above 98.5%, which outperforms the reported 2D membranes. Characterization and simulation results show that the introduction of hyaluronate‐grafted β‐cyclodextrin not only expands the interlayer channels of GO membranes but also enables the membranes to operate stably under harsh conditions with the help of host–guest interactions. This universal supramolecular assembly strategy provides new opportunities for the preparation of 2D membranes with high separation performance and reliable and stable nanofluidic channels.