Mesoporous materials based on lyotropic liquid crystal templates with precisely defined and flexible nanostructures offer an alluring solution to the age-old challenge of water scarcity. In contrast, polyamide (PA)-based thin-film composite (TFC) membranes have long been hailed as the state of the art in desalination. They grapple with a common trade-off between permeability and selectivity. However, the tides are turning as these novel materials, with pore sizes ranging from 0.2 to 5 nm, take center stage as highly coveted active layers in TFC membranes. With the ability to regulate water transport and influence the formation of the active layer, the middle porous substrate of TFC membranes becomes an essential player in unlocking their true potential. This review delves deep into the recent advancements in fabricating active layers using lyotropic liquid crystal templates on porous substrates. It meticulously analyzes the retention of the liquid crystal phase structure, explores the membrane fabrication processes, and evaluates the water filtration performance. Additionally, it presents an exhaustive comparison between the effects of substrates on both polyamide and lyotropic liquid crystal template top layer-based TFC membranes, covering crucial aspects such as surface pore structures, hydrophilicity, and heterogeneity. To push the boundaries even further, the review explores a diverse array of promising strategies for surface modification and interlayer introduction, all aimed at achieving an ideal substrate surface design. Moreover, it delves into the realm of cutting-edge techniques for detecting and unraveling the intricate interfacial structures between the lyotropic liquid crystal and the substrate. This review is a passport to unravel the enigmatic world of lyotropic liquid crystal-templated TFC membranes and their transformative role in global water challenges.