A three-dimensional (3D) biomimetic SERS substrate with hierarchical nanogaps was formed on the bioscaffold arrays of cicada wings by one-step and reagents-free ion-sputtering techniques. This approach requires a minimal fabrication effort and cost and offers Ag nanoislands and Ag nanoflowers with four types of nanogaps (<10 nm) on the chitin nanopillars to generate a high density of hotspots (∼2000/μm2). The 3D biomimetic substrate shows a low detection limit to Rhodamine 6G (10(-13) M), high average enhancement factor (EF, 5.8×10(7)), excellent signal uniformity (5.4%), good stability, and suitability in biosensing. Furthermore, the finite-difference time-domain (FDTD) electric-field-distribution simulations illustrate that the 3D biomimetic SERS substrate provides the high-density hotspot area within a detection volumem, resulting in enormous SERS enhancement. In addition, the conspicuous far-field plasmon resonance peaks were not found to be a strong requirement for a high EF in 3D biomimetic substrates. Additionally, the novel substrate was applied in label-free animal viruses detection and differentiation with small amounts (1.0 μL) and low concentrations of analyte (1×10(3) PFU/mL), and it exhibited potential as an effective SERS platform for virus detection and sensing.