Natural DNA was introduced to thin layer chromatography (TLC) with an aim to separate chemicals like DNA-affinity compounds and enantiomers. By cross-linking polyvinyl alcohol (PVA) with glutaraldehyde (GA) and subsequent cross-linking DNA with a UV irradiation, a DNA/PVA interpenetrating polymer network (IPN) is formed and was used to coat the surface of the porous silica particles of the TLC. Three typical DNA-binding compounds and eight amino acid enantiomers were used as model chemicals to investigate the chromatographic behavior of the modified TLC, and high separation efficiency was observed in both classes of the chemicals. On the practical side, the DNA-modified TLC have high prospects in diverse applications, including efficacy evaluation of a medicine, toxicity assessment of a pollutant at the molecular level, as well as separation of enantiomers such as dyes, amino acids, peptides, proteins, nucleotides, and drugs.