Supramolecular self-assembly of conjugated diacetylenic amphiphile-tethered ligands photopolymerize to afford polydiacetylene (PDA) functional liposomes. Upon specific interaction with a variety of biological analytes in aqueous solution, PDA exhibits rapid colorimetric transitions. The PDA nanoassemblies, which are excellent membrane mimics, include an ene–yne polymeric reporter responsible for the chromatic transitions and the molecular recognition elements that are responsible for selective and specific binding to the biological target. A bottleneck in the fabrication of these colorimetric biosensors is the preparation of the diacetylenic monomer embedded with the recognition element of choice. In the present work, we make use of copper-catalyzed azide–alkyne cycloaddition (CuAAC) as key step in the preparation of sugar-coated liposome biosensors. The regioselective click ligation of the triacetylenic N-(2-propynyl)pentacosa-10,12-diynamide (NPPCDAM) with a variety of mannose- and lactose-tethered azides afforded chemo- and regioselectively the corresponding 1,2,3-triazole. The obtained diacetylenic monomers were incorporated efficiently into vesicles to afford functional mannose- and lactose-coated glycoliposomes. The obtained PDA-based click glycoliposomes have been characterized by using transmission electronic microscopy (TEM), dynamic light scattering (DLS), and UV/Vis spectroscopy. The efficiency of the reported approach was demonstrated by the rapid optimization of the hydrophilic spacer between the lipidic matrix and the mannose head group for the colorimetric detection of Concavalin A.