The feasibility and sensitivity of optical waveguide lightmode spectroscopy (OWLS) in studying DNA-protein interaction are dissused. In the label-free DNA and DNA-protein interaction detection assay based on optical waveguide lightmode spectroscopy (OWLS), the probe was located online onto the amino silanized sensor chip of optical waveguide lightmode spectroscopy by glutaraldehyde covalent coupling; the DNA-protein complex, combination of NF-κB p50 homodimer and oligonucleotide with binding site to NF-κB p50 homodimer and binding sequence to probe, was used as the detect molecule. The different concentrations of p50 in detection samples and affinity of oligonucleotides with different binding site sequence of NF-κB to p50 were tested in this system. With this detection system, as low as 0.33 nmol/l of NF-κB p50 in the samples was detected, and the affinity of oligonucleotides with different binding site sequence to NF-κB is quite different. The label-free studying system of DNA-protein interaction based on the optical waveguide lightmode spectroscopy technique is feasible and high sensitivity, and this system can be used in the research targeted at the interaction of DNA and DNA-binding proteins and detection of the affinity of oligonucleotides to binding protein.