Current metabolomic studies of ischemic brain mainly attach importance on a certain ischemic period, are lack of data about dynamic metabolites in ischemic stroke process, especially early period. Thus, in this study, (1)H NMR spectroscopy was used to investigate biochemical changes in the early stages of rats' focal cerebral ischemia reperfusion (I/R) injury. Serum samples of 0, 0.5, 1, 3, 6, 12, 24 h of reperfusion, based on multivariate data analyses, were tested to analyze the changing of metabolites during the early disease process. Partial least squares-discriminant analysis scores plots of the (1)H NMR data revealed clear differences among the experiment groups. Combination the results of loading plot and t-test, we found that 13 metabolites were changed significantly. Among that, malonic acid and glycine are the most noticeable variable metabolites. Dramatic changed malonic acid and glycine most probably served as biomarkers in this study. These findings help us understand the biochemical metabolite changes in early ischemic stroke stages, especially different periods. That may be conducive to distinguish at-risk individuals, benefit early diagnosis and understand the dynamic pathogenesis of early cerebral ischemia.