The interaction between farrerol and calf thymus DNA in a pH 7.4 Tris-HCl buffer was investigated with the use of neutral red (NR) dye as a spectral probe by UV-vis absorption, fluorescence, and circular dichroism (CD) spectroscopy, as well as viscosity measurements and DNA melting techniques. It was found that farrerol molecules could intercalate into the base pairs of DNA as evidenced by decreases in iodide quenching effect and single-stranded DNA (ssDNA) quenching effect, induced CD spectral changes, and significant increases in relative viscosity and denaturation temperature of DNA. Furthermore, the spectral data matrix of the competitive reaction between farrerol and NR with DNA was resolved with an alternative least-squares (ALS) algorithm, and the concentration profiles in the reaction and the corresponding pure spectra for three species (farrerol, NR, and DNA-NR complex) were obtained. This ALS analysis demonstrated the intercalation of farrerol to the DNA by substituting for NR in the DNA-NR complex. Moreover, the thermodynamic parameters enthalpy change (ΔH°) and entropy change (ΔS°) were calculated to be -16.49 ± 0.51 kJ mol(-1) and 32.47 ± 1.02 J mol(-1) K(-1) via the van't Hoff equation, which suggested that the binding of farrerol to DNA was driven mainly by hydrophobic interactions and hydrogen bonds.