Vanillin (VAN) and ethyl vanillin (EVA) are widely used food additives as flavor enhancers, but may have a potential security risk. In this study, the properties of binding of VAN or EVA with calf thymus DNA (ctDNA) were characterized by multi-spectroscopic methods, multivariate curve resolution-alternating least-squares (MCR-ALS) algorithm and molecular simulation. The concentration profiles for the components (VAN or EVA, ctDNA and VAN-ctDNA or EVA-ctDNA complex) by the MCR-ALS analysis showed that VAN or EVA interacted with ctDNA and formed VAN-ctDNA or EVA-ctDNA complex. The groove binding of VAN or EVA to ctDNA was supported by the results from viscosity measurements, melting studies, denaturation experiments, and competitive binding investigations. Analysis of the Fourier transform infrared spectra corroborated the prediction by molecular docking that VAN and EVA preferentially bound to thymine bases region of ctDNA. The circular dichroism and DNA cleavage assays indicated that both VAN and EVA induced conformational change (from B - like DNA structure toward to A - like form), but didn't lead to a significant damage on DNA. The fluorescence quenching of Hoechst 33,258-ctDNA complex by VAN or EVA was a static quenching, and hydrogen bonding and van der Waals forces were main forces. This study has provided insights into the mechanism of interaction between VAN or EVA with ctDNA, and may also help better understand their potential toxicity with regard to food safety. Graphical Abstract VAN or EVA binds to A-T rich regions of ctDNA in the minor groove.