The identification of new biomarkers (e.g., metabolic biomarkers) will facilitate not only the diagnosis of stroke but also the differentiation of stroke subtypes, especially the discrimination of ischaemic stroke from intracerebral hemorrhage. Herein, we develop for the first time an ultra-high-pressure liquid chromatography tandem mass spectrometry (UHPLC-MS)-based targeted metabolomic method to screen the metabolic biomarkers of stroke and identify the fatty acid metabolite 20-hydroxy-leukotriene B4 (20-OH-LTB4) and its key enzyme cytochrome P450 family 4 subfamily F member 2 (CYP4F2) as the potential biomarkers for differentiating healthy persons, acute ischemic stroke (AIS) patients, and intracerebral hemorrhage stroke (ICH) patients. We evaluated 158 fatty acids and their metabolites in 177 serum samples obtained from 65 healthy volunteers, 70 AIS patients and 42 ICH patients, and identified the potential biomarkers associated with ICH by using multivariate statistical analysis. We found that 20-OH-LTB4 and arachidonic acid can be used to discriminate ICH patients from healthy individuals, and 20-OH-LTB4 and 17, 18-epoxy-eicosatetraenoic acid (7,18-EpETE) can be used to differentiate the subtypes of ICH and AIS. Especially, 20-OH-LTB4 may function as a potential biomarker for ICH diagnosis and risk assessment, and it can discriminate ICH patients from healthy individuals and AIS patients. Moreover, we identified CYP4F2 protein as a potential biomarker of ICH for prevention and treatment assessment. This method may provide a powerful platform for ICH diagnosis, prevention, and treatment assessment.