Nature possesses a powerful ability to assemble multiple complex structures to fabricate hierarchical biological structures in a living-assembled way. However, it is still a huge challenge for artificial systems to fabricate and characterize hierarchical living assemblies with well-defined and controllable but complex structures. In this work, we proposed a new concept for the fabrication of multiblock fluorescent microcolumns, which relies on the cooperation between the controllable host–guest complexation based on cucurbituril (CB) and the living assembly of nanotubular supramolecular polymers composed of CB and NaBr in aqueous solution. By using the complexation of CB with different guest numbers of luminogens with aggregation-induced emission (AIEgens) characteristics, and the difference in affinity between CB and different types of AIEgens, the concentration-controlled and self-sorting-controlled sequential living assembly are realized, respectively. Correspondingly, multiblock fluorescent microcolumns with different fluorescence emission are fabricated, and the molecular structure of each fluorescent block is analyzed by single crystal X-ray diffraction measurement. In addition, the living assembly of multiblock fluorescent microcolumns is visualized, understood, and regulated with the aid of AIEgens. The method developed here is expected to be extended to more guest molecules of CB and also provides a referential crystallization method for CB-based complexes.