A thin-film polyamide layer was synthesized on three different hollow-fiber support layers, each of which possess different morphological structures (i.e., sponge-like structures (SM-1), finger-like macrovoid close to the outer surface (SM-2), and middle-partitioned finger-like structures (SM-3)). In this work, we found that forward osmosis (FO) performance of thin-film composite (TFC) membranes could be significantly affected by their sub-layer morphology. During FO tests, SM-1 showed the highest water permeability of 0.742 LMH/bar followed by SM-3 and SM-2. In addition, SM-1 exhibited the lowest solute permeability of 0.039 LMH, followed by SM-2 and SM-3. To more systematically evaluate the effect of the support layer structures on the performance of TFC FO membranes, the structural parameters (S) of the membranes were estimated using the dusty gas model (DGM) prior to interfacial polymerization on the support layers. The S values of the support layers obtained from the DGM method are in accord with the FO membrane performance. Although the conventional S parameter determination method showed relatively similar values with the DGM method, the overall trend corresponds, confirming that the smaller S value is related to a smaller internal concentration polarization decrease. Moreover, this study clearly demonstrates that the support layer needs to be considered alongside the active layer of TFC FO membranes, and that a sponge-like support layer can be a better option for high-performance FO TFC membranes.