To design a bio-sensor for DNA detecting, we proposed a polarization free transmission type guided mode resonance biosensor with high sensitivity in visible wavelength was proposed for DNA detection with the FDTD software simulation based on the rigorous coupled wave theory and equivalent theory. When ssDNA was detected, the resonance wavelength was 481.46 nm. When dsDNA was detected, the resonance wavelength was 490.11 nm. When no DNA is present in the detection area, no transmitted light appears. Hence, it can be used to distinguish between single and double-stranded DNA. This bio-sensor can maintain the biological activity of the sample and avoid physical or chemical damage of the sample, which is convenient for multiple analysis and detection, and owns potential applications in the field of biological detection.