The present study sought to evaluate the interaction between aflatoxin G1 and free DNA in vitro through different analytical techniques. The UV-visible spectra results showed that the structure of DNA might be changed with a new aflatoxin G1-DNA complex forming, which indicated that the interacting mode between them was the intercalating mode. The DNA melting temperature increased by 12.80 °C, suggesting that the DNA double helix structure was more compact and stable through intercalation. The circular dichroism (CD) spectra results indicated that the interaction of aflatoxin G1 with DNA induced the DNA base stacking changes. The results of agarose gel electrophoresis and fluorescence microscope further verified that the interacting mode between aflatoxin G1 and DNA was intercalation mode. According to the fluorescence spectrum data, the binding constant was calculated 6.24 × 104 L·mol-1. The thermodynamic results demonstrated that the reaction of aflatoxin G1 intercalating to DNA was a spontaneous reaction. The elimination results suggested that aflatoxin G1 could be enriched and removed by DNA intercalation through magnetic beads separation, with the removal efficiency of 93.73%. The study results would provide a theoretical basis for establishing a new aflatoxin removal method based on DNA intercalation.