Rapid diagnosis and timely treatment improves the outcome in patients with ischemic stroke, but a rapid and sensitive blood test for ischemic stroke does not exist. This study tested whether a panel of biomarkers might be useful in the diagnosis of acute ischemic stroke. Consecutive patients with suspected stroke presenting to the emergency department of a university hospital in Korea were enrolled. Plasma specimens were assayed for brain natriuretic peptide, D-dimer, matrix metalloproteinase-9, S100β, and a proprietary composite multimarker index (MMX). There were 139 patients in this study, 89 of whom were diagnosed with acute ischemic stroke, 11 with acute cerebral hemorrhage, and 39 with other brain disorders. The MMX value was significantly higher in the patients with acute ischemic stroke in comparison to 57 healthy controls (p <0.001), but there was no significant difference between the MMX value in patients with acute ischemic stroke vs those with acute cerebral hemorrhage (p = 0.884). The discriminatory capacity of MMX was modest, with an area under the receiver-operating-characteristic curve of 0.714 for acute stroke. Ischemic stroke was not diagnosed by any of the biochemical markers individually. Although the data suggest that MMX may be helpful to diagnose an acute stroke, it does not discriminate between acute ischemic stroke and acute hemorrhagic stroke.