Interlayered thin-film nanocomposite (TFN) membranes have shown the potential to boost nanofiltration performance for water treatment applications including the removal of organic micropollutants (OMPs). However, the effects of substrates have been overlooked when exploiting and evaluating the efficacy of certain kinds of interlayers in tailoring membrane performance. Herein, a series of TFN membranes were synthesized on different porous substrates with identical interlayers of metal-organic framework nanosheets. It was revealed that the interlayer introduction could narrow but not fully eliminate the difference in the properties among the polyamide layers formed on different substrates, and the membrane performance variation was prominent in distinct aspects. For substrates with small pore sizes exerting severe water transport hindrance, the introduced interlayer mainly enhanced membrane water permeance by affording the gutter effect, while it could be more effective in reducing membrane pore size by improving the interfacial polymerization platform and avoiding PA defects when using a large-pore-size substrate. By matching the selected substrates and interlayers well, superior TFN membranes were obtained with simultaneously higher water permeance and OMP rejections compared to three commercial membranes. This study helps us to objectively understand interlayer efficacies and attain performance breakthroughs of TFN membranes for more efficient water treatment.