Abstract: Stroke is a kind of cerebrovascular disease with high disability rate and high mortality rate, it is divided into two subtypes of ischemic stroke and hemorrhagic stroke, which have similar clinical symptoms, but the treatment strategies are completely different. At present, the diagnoses mainly rely on medical imaging technology, but it is time-consuming, costly and difficult to distinguish the two subtypes of stroke, which affects the treatment and prognosis of the disease. In this study, 158 fatty acid metabolites in plasma from 59 healthy volunteers, 60 patients with acute cerebral ischemia and 36 patients with intracerebral hemorrhage were analyzed by ultra-high performance liquid chromatography-mass spectrometry (UPLC-MS), and the data was investigated by multivariate statistical analysis and receiver operating characteristic curve. The results showed that fatty acid metabolites can effectively distinguish healthy people from stroke patients, and can distinguish between acute cerebral ischemia and intracerebral hemorrhage. Thus, fatty acid metabolites are promising potential diagnostic biomarker for stroke. The results provide a new basis for the study of pathogenesis and diagnostic markers of stroke.