In this study, we developed a systematic evolution of ligands by exponential enrichment (SELEX) method using a combination of magnetic beads immobilization and flow cytometric measurement. As an example, the selection of streptavidin-specific aptamers was per-formed. In this protocol, the conventional SELEX pro-cedure was optimized, frst using magnetic beads for target immobilization to facilitate highly efficient separ-ation of the binding single-stranded DNA (ssDNA)aptamers from the unbound ssDNAs, and second using flow cytometry and fluorescein labeling to monitor the enrichment. The sensitivity of flow cytometry was ade-quate for ssDNA quantification during the SELEX pro-cedures. The streptavidin-specific aptamers obtained in this work can be used as tools for characterization of the occupancy of streptavidin-modified surfaces with biotinylated target molecules. The method described in the study is also generally applicable to target mol-ecules other than streptavidin.