Monomeric streptavidin (mSA) offers a combination of structural and binding properties that are useful in many applications, including a small size and monovalent biotin binding. Because mSA contains a structurally important disulfidebond, the molecule does not fold correctly when expressed inside the cell. We show that mSA can be expressed in a functional form in Escherichia coli by fusing the OmpA signal sequence at the amino terminus. Expressed mSA is exported to the periplasm, from which the molecule leaks to the medium under vigorous shaking. Purified mSA can be conjugated with FITC and used to label microbeads and yeast cells for analysis by flow cytometry, further expanding the scope of mSA-based applications. Some applications require recombinant fusion of mSA with another protein. mSA fused to EGFP cannot be secreted to the medium but was successfully expressed in an engineered cell line that supports oxidative folding in the cytoplasm. Purified mSA-EGFP and mSA-mCherry bound biotin with high affinity and were successfully used in conventional flow cytometry and imaging flow cytometry. Finally, we demonstrate the use of mSA in ELISA, in which horseradish peroxidase-conjugated mSA and biotinylated secondary antibody are used together to detect primary antibody captured on an ELISA plate. Engineering mSA to introduce additional lysine residues can increase the reporter signal above that of wild-type streptavidin. Together, these examples establish mSA as a convenient reagent with a potentially unique role in biotechnology.