Creation of new two-dimensional (2D) architectures has attracted significant attention in the field of selfassembly for structural diversity and new functionalization. Although numerous 2D polymer nanosheets have been reported, 2D nanosheets with tubular channels have been unexplored. Herein, we describe a new strategy for the fabrication of stimulus-responsive conjugated polymer 2D nanosheets with hollow cavities. Amphiphilic macrocyclic diacetylenes self-assembled in an aqueous solution in a columnar manner to afford bilayered 2D nanosheets with intrinsically tubular nanochannels. UV-induced polymerization resulted in the generation of blue-colored tubular conjugated polydiacetylene 2D nanosheets. Immobilization of gold nanoparticles, fluorescence labeling with FRET phenomenon and colorimetric DNA sensing were demonstrated with these new 2D nanosheets. In addition, the free NH 2 containing polymerized 2D nanosheet was utilized for conductivity behavior and grafting on graphene oxide (GO).