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The World Health Organization has recently introduced molecular prognostic-diagnostic biomarkers in the classification of Central Nervous System (CNS) tumors. In order to characterize subclasses of tumors that cannot find a precise location in the current classification, and, or cannot be tested because of scant material, it is important to find new molecular biomarkers in tissue and, or biological fluid samples. In this study, we identified serum microRNAs that could serve as biomarkers for the diagnosis and prognosis of patients with tumors of glial origin. We retrospectively analyzed microRNA expression in the serum extracellular vesicles of patients with tumors of glial origin. Extracellular vesicles RNA was analyzed by Nanostring. qRT-PCR confirmed 6 overexpressed microRNAs: hsa-miR-4443, hsa-miR-422a, hsa-miR-494-3p, hsa-miR-502-5p, hsa-miR-520f-3p, and hsa-miR-549a. Hsa-miR-4443 was the only microRNA that showed significant differences in most comparisons. In situ hybridization (ISH), confirmed that our signature was mostly expressed in cancer cells. Importantly, hsa-miR-549a and hsa-miR-502-5p expression predicted prognosis in patients with tumors of glial origin. Although more studies are needed, we demonstrated that serum vesicles microRNA profiles are promising diagnostic and prognostic molecular biomarkers that will find an actual application in the clinical practice of CNS tumors., Highlights • We identified a microRNA signature in the serum extracellular vesicles of patients with glial tumors. • The signature was differentially expressed among tumors and controls, showing potentials in the diagnosis of Gliomas. • Hsa-miR-549a and hsa-miR-502-5p expression predicted prognosis in patients with tumors of glial origin. Research in context To find targeted therapies and patients sensitive to specific treatments, we need to better classify different groups of brain tumors identifying novel molecular biomarkers. The most common brain tumors are Gliomas. In this article, we identified a group of very short genes (microRNAs) in the extra-cellular vesicles of the serum of 8 normal and of 28 patients with gliomas. The expression of such genes differs among the different groups of tumors and normal patients, and can help in the diagnosis of gliomas. Interestingly, two microRNAs could predict the outcome of the disease and might find future clinical applications.