Stimuli-responsive hydrogel microfibers and microtubes are in great demand for biomedical applications due to their similarity to the native extracellular matrix. In this study, we prepared pH- and temperature-responsive hydrogel microfibers and microtubes using a microfluidic device through alginate-templated photopolymerization. Hydrogel monomer solutions containing N-isopropylacrylamide (NIPAm) and sodium acrylate (SA) or allyl amine (AA) were irradiated with UV light to invoke in situ photopolymerization. A repulsive force between the ionized SA or AA groups caused by protonation/deprotonation of the acrylate or amine groups, respectively, led to changes in the diameters and wall thicknesses of the fibers and/or tubes depending on the pH of the medium. Poly(NIPAm) is a well-known thermally responsive polymer wherein the NIPAm-based copolymer microfibers exhibited a thermal behavior close to the lower critical solution temperature. We have demonstrated that these multistimuli-responsive volume changes are fully reversible and repeatable. Furthermore, the positively charged microfibers were shown to exhibit cell adhesion, and the number of cells attached to the microfibers could be further increased by supplying nutrients, presenting the possibility of their application in tissue engineering and other biomedical fields.