By utilizing multispectrosopic techniques, the toxic interaction of 2-aminoanthraquinone (2-AAQ) with calf thymus deoxyribonucleic acid (ctDNA) was investigated in vitro under simulated physiological conditions. The experimental results proved that 2-AAQ has a toxic interaction with ctDNA. The binding capacity of DNA with 2-AAQ is diminishing as the pH value of system increasing in the optimization of experimental condition. Moreover we selected pH 7.4, which is nearly physiological condition to enhance the practical significance. According to the Stern-Volmer equation, the quenching was the static quenching process. And the quenching constant Kq can be derived from the fluorescence quenching spectrogram. Ultraviolet absorption spectra and the change in the fluorescence intensity at different ionic strengths further indicated that there was electrostatic binding between 2-AAQ and ctDNA. The circular dichroism experiment showed that the DNA conformation varied from B to A conformation. The basic group enhanced after 2-AAQ embedding. The double helix is more compact, and the DNA conformation changes.