Hydrogel-based sensory structures were developed by embedding polydiacetylene supramolecules into poly-(ethylene glycol)-diacrylate (PEG-DA) to detect chemical gases and cyclodextrin and to determine pH values on the basis of a fluorescence change. We found the optimal condition for patterning-fabrication by controlling the volumetric mixture ratio of the water-soluble PEG-DA and aqueous polydiacetylene vesicle solution. Then, we determined that this hydrogel-based polydiacetylene structure optically responded selectively against vapor-phase targets: ammonia, ethanol, and aldehyde; aqueous solutions with various pH values; and cyclodextrin derivatives. These results could be extended to various label-free sensing applications of hydrogel-based chemo-biosensors.