5-羧基荧光素是制备水解稳定荧光共轭物的有用试剂，也是合成其它荧光素衍生试剂的有用起始材料。它是通过苯三羧酸酐与间苯二酚和氯化锌反应制备的5-和6-异构体的混合物。尽管这些同分异构体具有几乎相同的性质，但当与其他分子结合时，在共轭极性、内部荧光猝灭以及相关罗丹明标记试剂的情况下，已观察到标记特异性的差异。此外，荧光素衍生物的多步合成制备需要具有异构体纯度的起始原料，以便反应产物易于纯化和鉴定。
5-FAM contains a carboxylic acid that can be used to react with primary amines via carbodiimide activation of the carboxylic acid. Fluorescein is the most common fluorescent derivatization reagent for labeling biomolecules. In addition to its relatively high absorptivity, excellent fluorescence quantum yield, and good water solubility, fluorescein has an excitation maximum that closely matches the 488 nm spectral line of the argon-ion laser.