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 performed. In this protocol, the conventional SELEX procedure was optimized, first using magnetic beads for target immobilization to facilitate highly efficient separation 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 adequate for ssDNA quantification during the SELEX procedures. 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 molecules other than streptavidin.