Point‐of‐care testing (POCT) of clinical biomarkers is critical to health monitoring and timely treatment, yet biosensing assays capable of detecting biomarkers without the need for costly external equipment and reagents are limited. Blood‐based assays are, specifically, challenging as blood collection is invasive and follow‐upprocessing is required. Here, we report a versatile assay that employs hydrogel microneedles (HMNs) to extract interstitial fluid (ISF), in a minimally invasive manner integrated with graphene oxide‐nucleic acid (GO.NA)‐based fluorescence biosensor to sense the biomarkers of interest in situ. The HMN‐GO.NA assay is supplemented with a portable detector, enabling a complete POCT procedure. Our system could successfully measure four clinically important biomarkers (glucose, uric acid (UA), insulin, and serotonin) ex vivo, in addition, to accurately detecting glucose and UA in vivo.