The quaternary isoquinoline alkaloid, sanguinarine (SG) exhibits a wide range of biological activities. This study examines spectral changes expected from SG binding to proteins. Fluorescence spectra of the cationic form of sanguinarine (SG(+)) are sensitive to environment polarity. On the other hand, spectra of the neutral form of sanguinarine, pseudobase (SGOH) and dihydrosanguinarine (DHSG, the first metabolite of SG) exhibit higher sensitivity to the ability of solvent to form a solute-to-solvent hydrogen bonds. Interaction with cysteine has been the only mode of SG binding to enzymes that has been considered so far. In reality, our experiments have revealed spectral changes on specific interactions of SG(+) with Cys, Glu and Tyr in the protic environment and with Arg and Glu in the aprotic environment. We have also detected interactions of SGOH with Cys in the protic environment and with Cys, Glu and Lys in the aprotic environment. The DHSG spectra were only altered in the presence of the Cys analog in the protic environment. We have also demonstrated that spectral change analysis can aid investigation of SG/DHSG interactions with proteins and we were able to identify SG(+)-binding site on Na(+)/K(+)-ATPase.