A novel biopolymer, deoxyribonucleic acid-hexadecyltrimethylammonium chloride (DNA-HCTAC), is used as the core layer material in optical waveguide, and the cleanroom technology is successfully applied to fabricate the single-mode channel waveguides with low propagation loss. The prepared DNA-HCTAC material shows high optical quality at the optical telecommunication wavelengths, such as high transparency, relatively high refractive index and low birefringence. In the fabrication approach, polymethyl methacrylate (PMMA) is used as a barrier layer to protect the DNA-HCTAC material from the corrosive of photoresist developer, and the etching conditions are optimized to form the smooth wall and sharp cross-section of the waveguide. Lastly, the optical characteristics of DNA-HCTAC channel waveguides are measured. The results show that the DNA-HCTAC waveguide operates with single-mode propagation and has a low optical loss.