Stroke is one of the leading causes of disability and death among adults worldwide and results in numerous biochemical alterations. However, few efficient biomarkers are clinically available to diagnose stroke because of the limitations of biomarkers and their probes. In this work, we utilized frozen brain slices of middle cerebral artery occlusion (MCAO) in a mouse model of ischemia to select a specific binding aptamer, termed LCW17, by tissue-based SELEX (systematic evolution of ligands by exponential enrichment). LCW17 was enhanced in binding in ischemic brain slices compared to sham control. We identified the binding target of LCW17 as vigilin. Vigilin is increased in ischemia brain slices and exhibits enhanced release from cultured hippocampal neurons after oxygen glucose deprivation in vitro. Taken together, ischemic brain slice-based aptamer selection will enable identification of more probes and potential target molecules for diagnosis and therapy of ischemic stroke. Aptamer LCW17 and vigilin may potentially be applied to define the molecular mechanism underlying ischemic stroke, as well as its diagnosis.