A water-soluble two-dimensional supramolecular organic framework (2D SOF) was prepared via self-assembly of cucurbit[8]uril (CB[8]) and a three-arm flat linker molecule, which contains a benzene ring as the core and three Brooker's merocyanine (BM) analogs as arms. The strong host–guest interactions between BM and CB[8] and the directional head-to-tail stacking modes between the BM arms synergistically led to the formation of a 2D SOF. The structure of the 2D SOF was verified by ¹H NMR, 2D ¹H NMR NOESY, and DLS characterizations, while the monolayer structure was characterized by Cryo-TEM and AFM measurements. The 2D SOF exhibited an obvious AIE enhancement effect in H₂O. In addition, DNA induced photoluminescence enhancement was observed for the monomer. As a result, this AIEgen-based 2D SOF could feature not only as a cell visualizer but also as a tracker for the nucleus in biological imaging due to the dynamic assembly process.