<p>Although continuous glucose monitoring (CGM) is essential for precise and personalized diabetes management, conventional approaches rely on invasive finger-prick blood sampling. Furthermore, existing CGM systems suffer from major limitations, including user discomfort, biofouling of implanted sensors, and unstable sensing components. To address these challenges, we present a hollow microneedle-based (MN) biosensor that minimally invasively accesses the interstitial fluid (ISF) for in-situ glucose monitoring. The MN biosensor integrates a miniaturized three-electrode system within the lumen of MNs filled with synthesized vacancy-regulated Prussian blue intercalated thermoplastic graphite composite (GP@PB). The GP@PB composite, prepared through a precipitation-conversion strategy, exhibits hierarchical and hollow morphology that provides a large active surface area and mitigates structural degradation. The sensing interface is further protected by an external MN body and a poly(methyl methacrylate) (PMMA) substrate, both of which greatly improve mechanical robustness and electrochemical stability. The MN biosensor enables real-time and continuous glucose monitoring in ISF with great sensitivity, selectivity, biocompatibility, and long-term reliability. In vivo studies in rat models validate the real-world feasibility of the biosensor by providing dynamic analysis of ISF glucose in response to metabolic variations, showing a strong correlation with gold-standard results measured using commercial devices. This work facilitates the clinical translation of minimally invasive CGM in personalized diabetes management, highlighting the potential of wearable electronics toward chronic disease healthcare.</p>