Label-free Surface-enhanced Raman spectroscopy (SERS) methods have great potential for detecting analytes of various sizes and dimensions with high sensitivity and selectivity. To mitigate the impact of interfering substances, SERS-active substrates with size-matched selectivity were designed by coating Au nanoparticles (Au NPs) on ZrO2 multilayer nanofibers (Au NPs/fZrO2) and referencing with Au NPs embedded on ZrO2 nanobowl (Au NPs/pZrO2). The detection efficiency study covered four small pesticide molecules, two live SARS-CoV-2 virus variants (Alpha and Delta), and three interfering substances. The results demonstrate that the fibrous structure of Au NPs/fZrO2 could effectively wet the sample and detect larger target molecules, such as live SARS-CoV-2 virus, thus improving efficiency by reducing unwanted molecules in the SERS signal. This substrate also showed high discrimination between Alpha and Delta variants. Au NPs/fZrO2 demonstrated similar trace detection capabilities to Au NPs/pZrO2 for pesticide molecules and virus variants; however, it particularly exhibited stronger peak intensities and more unique SERS peaks assigned to the variants. Thus, differences in substrate morphology affect the generation of hotspots and the distance between analyte and hotspots. These findings hold promise for the development of SERS-based label-free analytical method for trace detection of a variety of virus particles.