This bifunctional biotin conjugates demonstrates better binding and stronger fluorescence than biotin fluorescein. It has similar avidin-binding properties in terms of high affinity, fast association, and non-cooperative binding to avidin and streptavidin tetramers. These exceptional properties are attributed to the small size/length of the new ligand since all larger/longer biotin derivatives are known for their mutual steric hindrance and anti-cooperative binding in 4:1 complexes with avidin and streptavidin tetramers. Specific binding of this biotin-fluorescein conjugate towards avidin and streptavidin is accompanied by 84-88% quenching of ligand fluorescence.  It is used for the quantitation of biotin-binding sites. Both the fluorescence and absorbance of biotin-4-fluorescein are quenched upon binding to one of the four biotin-binding sites of streptavidin, avidin, or the streptavidin conjugates of fluorescent dyes or enzymes. As a result, when a known concentration of biotin-4-fluorescein is added to a known amount of streptavidin, one can estimate the number of biotin-binding sites.