DNA-based nano-biosensors are emerging scope in the field of biosensors. Many synthetic single stranded functional DNAs have been applied for development of such sensors, recently. Immobilization of DNA oligonucleotides on the surface of gold nanoparticles is a key step in the development of most colorimetric nano-biosensors. The bound DNA is usually thiolated and forms Au-S covalent bond to the surface of gold nanoparticles. To this endeavor, the DNA must get reduced prior to immobilization. There are a variety of approaches for reduction of thiolated DNAs that mostly employ Dithiothreitol (DTT). DTT-based DNA reduction is not always complete and the sulfhydryl DNA (DSH) is not the sole product of the reaction. The results of the reduction of a thiolated DNA with DTT is analyzed in this paper with high performance liquid chromatography (HPLC) in order to find an optimal condition for DTT based reduction. Finally, the optimal condition is compared to TCEP-based reduction for the efficiency of DNA immobilization on the surface of gold nanoparticles.