The interaction of a new derivative of thiazole orange (TO-3) with calf thymus DNA (ctDNA) has been investigated by fluorescence and absorption spectroscopy. When TO-3 binds to ctDNA, absorption bands exhibit significant hypochromicity at low base pair/dye ratio (BP/D ratio), and high BP/D show hyperchromicity with red shift. The spectral changes are attributed to the different species formed between TO-3 and ctDNA in the titration course of the dye molecule with DNA. Multivariate curve resolutions alternating least squares (MCR-ALS) is applied to the absorption measurements recorded to recover the concentration profiles and the pure spectra of the DNA/TO-3 complexes involved in the process. The binding constant and size of the binding site have been determined spectrophotometrically using the McGhee von Hippel equation. MCR-ALS has been used to reveal the precise concentration profiles of all detectable species formed between ctDNA and TO-3 and their pure spectral profiles.