Layer stacking of two-dimensional (2D) materials, such as graphene and transition metal dichalcogenides, is critical for controlling their physical and transport properties. By exploiting a specific stacking order, the electronic band structures of such 2D materials can be tuned, from which unusual and exotic properties may emerge. Graphene oxide (GO) undergoes layer stacking along with its photo- and thermal-induced reduction process; however, the underlying mechanism and dynamics during its layer stacking have not been revealed. In this study, we demonstrate time-resolved electron diffraction for monitoring the structural dynamics during the layer stacking of GO induced by ultraviolet photoexcitation. The experimental results accompanied by the density functional theory calculations reveal that AB stacking of graphitic domains of GO layers coincides within ∼40 ps with photoinduced removal of the epoxy-oxygen from the basal plane of GO via the strong interactions between the GO layers.