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Bis-ANS is a high-affinity fluorescent probe for nonpolar cavities in proteins. Its hydrophobic phenyl and naphthyl rings interact noncovalently with proteins and protein degradation products.

As with other anilinonaphthalene sulfonates (ANS), bis-ANS is essentially nonfluorescent in water but becomes noticeably fluorescent in a nonpolar environment. When free, bis-ANS has an excitation maximum at 390 nm and an emission maximum at 523 nm but undergoes a blue shift with an increase in fluorescence intensity when bound to protein.

Bis-ANS is frequently used to monitor the formation of protein aggregates and indicate protein folding and conformational changes. The dye is also used to detect Aβ fibers.