Plasmonic nanohole arrays have received significant attention, as they have highly advantageous optical properties for ultrasensitive and label-free biosensing applications. Currently, most of these subwavelength periodic apertures are mainly implemented on transparent materials, which results in multiple spectrally close transmission resonances. However, this spectral characteristic is not ideal for biosensing applications, as it complicates monitoring spectral variations. In this article, utilizing a hybrid substrate composed of a high refractive index dielectric interlayer over a transparent material, we show that gold nanohole arrays support spectrally isolated and well-defined plasmonic resonances that are easy to track. Compared to conventional configurations on transparent material, nanoholes on a hybrid substrate also exhibit plasmonic modes with well-preserved amplitudes, which is useful for reliable spectral monitoring. We show that nanohole arrays on a hybrid substrate are more sensitive to changes in surface conditions. Using a spectral integration method, which evaluates wavelength shifts in a large spectral window instead of monitoring only the plasmonic resonance wavelength, we obtain a detection limit as low as 2 x 10^(-5) RIU. Furthermore, we successfully demonstrate real-time monitoring of biomolecular binding interactions even at sub-1 ng/mL levels.