Responsive fluorescent microgels, that can selectively, reversibly, and rapidly convert the fluctuation in intracellular glucose level into fluorescence signal, have the potential use for intracellular glucometry to promote the understanding of physiology. Herein, we report one-pot synthesis of such a responsive fluorescent composite microgels, which is made of a representative apo-enzyme, apo-glucose oxidase (apo-GOx), interpenetrated in a composite gel network that is comprised of ZnO quantum dots covalently bonded onto crosslinked poly(ethylene glycol) dimethacrylate. The key of this one-pot synthesis is applying a high-temperature short-time heating (HTST) method, so that the naturally dynamic profile of apo-GOx can be maintained and harnessed on the composite microgels to allow the highly selective response to glucose over a glucose concentration range of 0–20 mM. While the composite microgels can undergo volume phase transitions and convert both an increase and a decrease in glucose concentration into fluorescence signal shortly (<1 s), the changes in average hydrodynamic diameter and fluorescence of the composite microgels can be fully reversible even after twenty cycles of adding/removing glucose, indicating a reversible and rapid time response to the glucose concentration variations. With the composite microgels as biosensors, the fluorescence of the composite microgels embedded in the model cancer cells B16F10 can be modulated in response to intracellular glucose level variations, which are derived from a change in glucose concentration in the culture medium by an external supply, or that can be triggered by biochemical reactions (with the β-galactosidase catalysed hydrolysis of lactose as a model reaction for achieving increased glucose levels, and the GOx catalysed oxidation of glucose for achieving decreased glucose levels).