Based on the reaction of acridine orange with deoxyribonucleic acid (DNA), and under the optimized condition, a novel method to determine the DNA was developed with the spectrophotometry. The absorbance of acridine orange at the maximum absorption wavelength of 444 nm responded to the concentration of DNA negatively with excellent linearity. It has an upper linear limit concentration of 8.0 microg x mL(-1) and a detection limit of 0.12 microg x mL(-1) with the correlation coefficient of 0.999 8. The method is simple, rapid and sensitive, and could be applied to sample assay satisfactorily. Finally, the reaction mechanism is discussed.