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The versatile design of stimuli-responsive microparticles embedding valuable biomolecules has great potential in a variety of engineering fields, such as sensors, actuators, drug delivery, and catalysis. Here, results are reported on thermoresponsive core–gap–shell (TCGS) microcapsules made of poly(N-isopropylacrylamide) (PNIPAm), which encapsulate hydrophilic payloads in a simple and stable manner. These are realized by a one-step microfluidic approach using the phase separation of a supersaturated aqueous solution of NIPAm. Various designs of the microcapsules are achieved by individual control of the swelling or by incorporating pH-responsive comonomers of the inner core and outer shell. The gap, i.e., the space between the inner core and outer shell, can be loaded with cargo-like nanoparticles. The outer shell can serve as a stimuli-responsive gateway for the transport of smaller molecules from the external solution. It is shown that the TCGS microcapsules are suitable as temperature controllable glucose sensors and hold promise in the design of controllable enzymatic reactions. The proposed platform provides an avenue for developing a new-generation of microparticles for diverse and efficient engineering applications.