<p>Hormones and metabolites jointly regulate physiology yet their dynamic interplay remains difficult to resolve due to the lack of technologies for continuous, multiplexed monitoring. Microneedle biosensors that access dermal interstitial fluid offer a minimally invasive approach to molecular profiling, but deployment is limited by trade-offs between fabrication scalability and precision, and by mechanical instability under skin deformation. Here we present a strain-isolated microneedle platform fabricated by combining two-photon polymerization with ultrasound-assisted moulding, enabling scalable replication of submicrometre-sharp microneedles (∼430-nm apex width) bearing hierarchical, high-surface-area microstructures. Nanostructured gold and platinum electrodes enable high-fidelity sensing at the single-needle level, providing a 4.7-fold higher peak current and a 9-fold larger electrochemical surface area, respectively. Mechanical decoupling of the sensing interface from tissue deformation preserves stable molecular access during motion. When integrated with battery-free wireless electronics and multiplexed aptameric and enzymatic sensors, the system enables continuous in vivo monitoring of serotonin and glucose for 12 h. In freely moving rats, the platform captures biomolecular dynamics associated with stress, feeding and circadian rhythms. This work establishes a mechanically robust, scalable microneedle biointerface for ambulatory molecular monitoring and context-aware assessment of physiological state.</p>