Abstract The binding behaviour of an azo dye, Allura Red AC with calf thymus deoxyribonucleic acid (ct-DNA) was investigated using UV-vis absorption spectroscopy, fluorescence quenching method, DNA melting and viscosity measurement. The physicochemical characteristics of Allura Red AC were investigated in 5 mM aqueous phosphate buffer of pH 7.0 at 25 °C. Spectrophotometric studies of the interaction between Allura Red A C and ct-DNA have shown that the binding constant was Kb=7.5×105 M-1 at 298 K. Thermodynamic parameters indicated that hydrogen bond and van der Waals play major roles in the interaction. A competitive binding with ethidium bromide (EB) showed that Allura Red AC displace EB from its binding site in ct-DNA. The thermal denaturation experiments show the melting temperature of ct-DNA increases (about 5.0 °C) due to binding of Allura Red AC. There is no obvious increasing of ct-DNA viscosity was observed. All of the experimental results show that the groove binding must be predominant.