Graphene quantum dots (GQDs) have received much research attention due to their unique optical/electrical/magnetic features. However, the intrinsic relationship between the structure and properties of GQDs remains not fully understood, because the controlled synthesis and the accurate structure determination of GQDs are still a great challenge. Here, single-layered GQDs are successfully obtained by the hydrothermal treatment of intercalated citrate in the confined space of 2D interlayer galleries of layered double hydroxides (LDHs). The distinct molecular structures of the single-layered GQDs reveal that the blue photoluminescence of the GQDs derives from a rigid π-conjugate plane structure, which is in accordance with theoretical calculations. This work will open the door towards a quantitative understanding of the structure-property effects of GQDs.