A microfluidic technique was employed to fabricate polydiacetylene (PDA)-embedded hydrogel microfibers. By taking advantage of calcium ion-induced insoluble hydrogel formation, supramolecularly assembled diacetylene (DA)-surfactant complexes were successfully immobilized in the calcium alginate fibers. Thus, instantaneous microfiber formation was observed when the core flow of DA supramolecules-containing alginate solution met the sheath flow of calcium ions. UV irradiation of the resulting fibers afforded blue colored PDAs, and the formation of a conjugated polymer was confirmed by heat-induced phase transition and by Raman spectroscopy. By adjusting the core and sheath flow rates, PDA-embedded hydrogel fibers of various sizes were obtained.