The interaction between heparin and acridine orange(AO) was studied by circular dichroism(CD). absorption and fluorescence spectra measurements. The strength of the dye-dye interaction (stacking tendency) between acridine orange cations bound to neighboring sites on the heparin was measured in terms of a ,stacking coefficient(K) and other method (β/α) derived from the spectrum of the bound dye. As the P/D molar ratio was increased, α band of at AO at 492nm was decreased while β bared at 470nm and γ band at 450nm were increased. The most stacking of AO was obtained at the P/D molar ratio of 25. An extrinsic optical activity was induced in the region corresponding to the absorption bands of AO when the dye became bound to the heparin. The magnitude of the molar ellipticity of heparin-AO complex was dependent on the P/D ratios. The conformational change of heparin occurred by forming complex with acridine orange.