The mechanism underlying the interaction of calf thymus DNA (ctDNA) with sesamol (SL), sesamin (SN), and asarinin (AN) was investigated by ultraviolet-visible (UV-Vis) and circular dichroism (CD) spectroscopies, DNA melting studies, and viscosity measurement under simulated physiological conditions (pH = 7.4). The results showed that ctDNA could produce the hypochromic effect for the UV absorbance peaks of the three compounds at 290 nm accompanied by a slight redshift; a higher concentration of ctDNA led to a greater hypochromic effect, and the binding constants of SL, SN, and AN to ctDNA were 2.65 × 104, 4.91 × 104, and 7.33 × 104 L/mol, respectively.The intensity of CD positive bands (276 nm) of ctDNA increased with the addition of the three compounds, and the negative bands (246 nm) were slightly reduced in intensity; a higher concentration led to a more significant change in the CD bands. With the presence of the three compounds, the melting temperatures of ctDAN were increased by 1.75, 2.51, and 6.63 ℃, respectively, and the viscosities of ctDNA were all increased to some extent. The results from the spectroscopic and viscosity experiments suggest that the interactions of SL, SN, and AN with DNA involve intercalation binding, and their interaction strengths were in the following order: AN > SN > SL.