Understanding the behavior of biomolecules on nanointerface is critical in bioanalysis, which is great challenge due to the instability and the difficulty to control the orientation and loading density of biomolecules. Here, we investigated the thermodynamics and kinetics of DNA hybridization on gold nanoparticle, with the aim to improve the efficiency and speed of DNA analysis. We achieved precise and quantitative surface control by applying a recently developed poly adenines (polyA)-based assembly strategy on gold nanoparticles (DNA-AuNPs). PolyA served as an effective anchoring block based on the preferential binding with the AuNP surface and the appended recognition block adopted an upright conformation that favors DNA hybridization. The lateral spacing and surface density of DNA on AuNPs can be systematically modulated by adjusting the length of polyA block. We found the stability of duplex on AuNP was enhanced with the increasing length of polyA block. When the length of polyA block reached to 40 bases, the thermodynamic properties were more similar to that of duplex in solution. Fast hybridization rate was observed on the diblock DNA-AuNPs and was increased along with the length of polyA block. We consider the high stability and excellent hybridization performance come from the minimization of the DNA-DNA and DNA-AuNP interactions with the use of polyA block. This study provides better understanding of the behavior of biomolecules on the nanointerface and opens new opportunities to construct high-efficiency and high-speed biosensors for DNA analysis.