In this study, nanopillar arrays of silicon oxide are fabricated through a process involving very-large-scale integration, for use as two-dimensional periodic relief gratings (2DPRGs) on silicon surfaces. Oligonucleotides are successively immobilized on the pillar surface, allowing the system to be used as an optical detector specific for the targeted single-stranded DNAs (ssDNAs). The surfaces of the oligonucleotides-modified 2DPRGs undergo insignificant structural changes, but upon hybridizing with target ssDNA, the 2DPRGs undergo dramatic changes in terms of their pillar scale. Binding of the oligonucleotides to the 2DPRG occurs in a way that allows them to retain their function and selectively bind the target ssDNA. The performance of the sensor is evaluated by capturing the target ssDNA on the 2DPRGs and measuring the effective refractive index (neff). The binding of the target ssDNA species to the 2DPRGs results in a color change from pure blue to red, observable by the naked eye along an angle of 15–20°. Moreover, effective medium theory is used to calculate the filling factors inside the 2DPRGs and, thereby, examine the values of neff during the structural changes of the 2DPRGs. Accordingly, these new films have potential applications as label-free optical biosensors.