We have developed new surface to ensure a proper spacing between immobilized biomolecules. While DNA microarray on this surface provided each probe DNA with ample space for hybridization with incoming target DNAs, the microarray showed enhanced discrimination efficiency for various types of single nucleotide polymorphism. The high discrimination efficiency holds for all tested cases (100:<1 for internal mismatched cases; 100:<28 for terminal mismatched ones). In addition, by investigating influence of hybridization temperature and washing condition on the fluorescence intensity and the discrimination efficiency with and without controlled mesospacing, it was observed that the nanoscale-controlled surface showed good discrimination efficiency in a wide range of temperature (37–50°C), and hybridization behavior on the surface was in agreement with the solution one. Intriguingly, it was found that washing process after the hybridization was critical for the high discrimination efficiency. For the particular case, washing process was so efficient that only 30 s washing was sufficient to reach the optimal discrimination ratio.