The interaction between astragalin (AST) from lotus leaf and deoxyribonucleic acid (DNA) in Tris-HCl buffer (pH=7.4) was investigated by the application of fluorescence spectroscopy and ultraviolet absorption spectroscopy, the effects of ionic strength and anion quencher KI on the fluorescence intensity of AST from lotus leaf and the system of AST-DNA were explored, and the competitive binding to DNA between AST from lotus leaf and Neutral Red(NR)dye was also studied. The results demonstrated that AST could bind to DNA and the formed complex quenched the intrinsic fluorescence of AST from lotus leaf through static quenching mechanism. The quenching rate constants of biomolecule(Kq)of the reaction of DNA with AST from lotus leaf were calculated to be 3.120×1012 and 2.630×1012 L?mol-1?s-1 by Stern-Volumer equation, the corresponding binding constants(Kd)were computed to be 3.412×104 and 1.762×104 L?mol-1 and the number of binding sites(n) was counted to be 1.007 and 0.962 between AST from lotus leaf and DNA at 298 and 308 K, respectively. When bound to DNA, the AST from lotus leaf showed hypochromic effect and red shift in the absorption spectra. It was also found that different ionic strength had little or no effect on the fluorescence intensity of AST and AST-DNA, but the fluorescence intensity of AST-DNA quenched by anionic quencher KI was much less than that of free AST. AST could be intercalated into DNA and displaced the NR from the NR-DNA complex. It was showed that AST from lotus leaf could combine with DNA in the mode of intercalation.