The two-dimensional (2D) materials offer atomic-level thickness and unique physical and chemical properties for the preparation of a new class of membranes, i.e., nanochannel membranes. The nanochannel membranes have been utilized in a broad spectrum of new separation applications. However, the instability of the nanochannels, interfacial instability of 2D materials, and the swelling problem could damage the membrane performance, such as permeability, selectivity, and service lifetime. Innovative strategies for constructing and regulating the nanochannels are enthusiastically explored to address these challenges. Along this line, in this work, we first provide insight into the mechanisms of the nanochannel construction, the separation mechanism, and the effect of nanochannels on the separation performance. Then, the strategies developed in the literature, in particular, the strategies for the preparation of ideal 2D nanosheets, the strategies for constructing nanochannels, and the strategies for regulating the characteristics of nanochannels (channel size, channel length, channel morphology, and channel surface physicochemical properties) are systematically summarized. After that, we briefly introduce the application of 2D-material-based nanochannel membranes and outline the current challenges and provide an outlook in the further exploration of separation mechanism, large-scale manufacturing, and the eventual commercialization of the membranes.