Rapid detection of brain natriuretic peptide (BNP) concentration can be used for the diagnosis of acute heart failure and for the evaluation of the effectiveness of a clinical therapy. We used the systematic evolution of ligands by exponential enrichment method to develop DNA aptamers for BNP whose sequences were determined by cloning method and consensus sequence analysis. A total of eight conserved sequences was identified. By combining the fluorescent-labeled aptamers with fast protein lab-on-chip analysis, we could achieve quantification of BNP concentrations with high speed, sensitivity, and specificity.