Hydrogel-based hollow microcapsules with good monodispersity and repeated glucose-response under physiological temperature and glucose concentration conditions have been fabricated by a simple emulsion-template approach. Double emulsions from microfluidic devices are used as templates to synthesize the monodisperse glucose-responsive microcapsules. In the poly(N-isopropylacrylamide-co-3-aminophenylboronic acid-co-acrylic acid) (P(NIPAM-co-AAPBA-co-AAc)) hydrogel shell of the microcapsules, the thermo-responsive PNIPAM network and the glucose-responsive AAPBA moiety are respectively used for actuation and glucose response, and the AAc moiety is used for adjusting the volume phase transition temperature of the shell. Glucose-responsive microcapsules prepared with 2.4 mol% AAc exhibit reversible and repeated swelling/shrinking response to glucose concentration changes within the physiological blood glucose concentration range (0.4–4.5 g L−1) at 37 °C. Rhodamine B and fluorescein-isothiocyanate-labeled insulin are used as model molecules and model drugs to demonstrate the potential application of the microcapsules for glucose-responsive controlled release. The microcapsules provide a promising and feasible model for developing glucose-responsive sensors and self-regulated delivery systems for diabetes and cancer therapy. Moreover, the microfluidic fabrication approach and research results presented here provide valuable guidance for the design and fabrication of monodisperse glucose-responsive microcapsules.