Stroke induces metabolic changes in the body, and metabolites have become potential biomarkers for stroke. However, the specific metabolites involved in stroke and the mechanisms underlying brain injury during stroke remain unclear. Surface-enhanced Raman spectroscopy (SERS) and liquid chromatography-mass spectrometry (LC‒MS) analysis of clinical serum samples from 69 controls and 51 ischemic stroke patients who underwent reperfusion within 24 hours were performed to identify differentially abundant metabolites. Mice were subjected to transient middle cerebral artery occlusion (tMCAO) and then intravenously injected with hypoxanthine. The infarct area was evaluated via tetrazolium chloride (TTC) staining, and behavior tests were conducted. Blood-brain barrier (BBB) leakage was assessed by Evans blue and IgG staining. Human blood vessel organoids were used to investigate the mechanism of hypoxanthine-induced pyroptosis of endothelial cells. SERS and LC‒MS revealed the metabolic profiles of serum from stroke patients and controls with high sensitivity, speed and accuracy. Hypoxanthine levels were significantly elevated in the acute stage of ischemic stroke in both patients and mice (p < 0.001 after Bonferroni correction). In addition, increasing hypoxanthine increased the infarct area and aggravated BBB leakage and neurobehavioral deficits in mice after ischemic stroke. Further mechanistic studies using endothelial cells, human blood vessel organoids, and stroke mice demonstrated that hypoxanthine-mediated gasdermin E (GSDME)-dependent pyroptosis of endothelial cells occurs through intracellular Ca overload. Our study identified hypoxanthine as an important metabolite that induces vascular injury and BBB disruption in stroke through triggering GSDME-dependent pyroptosis of endothelial cells.