Graphene oxide (GO) has been widely explored for the improvement of thin-film composite (TFC) membrane performance. However, the influences of GO flake lateral size on the polyamide (PA) TFC membrane properties and performances have not been investigated. In this study, GO suspensions with an average flake size ranging from 0.01 to 1.06 μm2 were prepared by varying the sonication duration between 0 and 8 h. The different sized GO flakes were embedded in the PA layer to examine the effect of their size on the morphology and performances of TFC forward osmosis (FO) membranes. The specific reverse solute flux and water flux of the GO-modified thin-film nanocomposite (TFN) membranes improved by over 60% and 50%, respectively, when the average GO flake size was reduced from 1.06 to 0.01 μm2 due to the formation of a thinner and more uniform PA layer. Large GO flakes deteriorated membrane performance by creating impervious regions that obstructed the reaction between monomers during the interfacial polymerization process resulting in defective PA layer formation. Whereas, smaller GO flakes distributed more uniformly in the PA layer creating fewer defects, and demonstrated better desalination performance and antifouling property than the TFN membranes modified with larger GO flakes. These results deliver strategies for future improvements in GO or 2D nanomaterial-based TFN membranes, where smaller flake size can be beneficial for minimizing PA layer defects.