Skin interstitial fluid (ISF) containing a great variety of molecular biomarkers derived from cells and subcutaneous blood capillaries has recently emerged as a clinically potential component for early diagnosis of a wide range of diseases; however, the minimally invasive sampling and detection of cell-free biomarkers in ISF is still a key challenge. Herein, we developed microneedles (MNs) that consist of gelatin methacryloyl (GelMA) and graphene oxide (GO) for the enrichment and sensitive detection of multiple microRNA (miRNA) biomarkers from skin ISF. The GO-GelMA MNs exhibited robust mechanical properties, fast sampling kinetics, and large swelling capacity, which enabled collecting ISF volume high to 21.34 μL in 30 min, facilitating effective miRNA analysis. It preliminarily realized the sensitive detection of three types of psoriasis-related miRNAs biomarkers either on the patch itself or in solution after release from the hydrogel by combining catalytic hairpin assembly signal amplification reaction. The automated and minimally invasive ISF miRNA detection technology of GO-GelMA MNs has great potential to monitor cell-free clinically informative biomarkers for personalized diagnosis and prognosis.