Tryptophan (Trp) residues within copoly(L-glutamic acid-co-L-Trp) (CAT) form a tricyclic structure when treatment with trifluoroacetic acid (TFA) and show reversible color change from yellow (above pH 8.0) to red (below pH 7.0) when the nitrogen atom in an indole ring dissociates positively in the neutral pH region. The interaction between TFA-treated GAT (GAT-T) and an anionic molecule such as warfarin was investigated using VIS and fluorescence spectrophotometry. Trp residues within GAT-T were found to interact with warfarin electrostatically besides hydrophobic interaction resulting in a color change from red to yellow. Release behavior of warfarin from GAT-T microspheres (MS) was also investigated. Binding amount and retention time of warfarin in GAT-T MS were found to be superior to those in untreated GAT MS.