In this paper, SiO2 support with 3D ordered nanohole structure was developed by using the nanopillars naturally created on the surface of the cicada wing as the template through a simple strategy of tetraethyl orthosilicate (TEOS) hydrolysis. Then 3D Ag/SiO2 substrate with a high density of hot spots was subsequently obtained by thermal deposition of Ag onto the SiO2 support. SEM and AFM observation reveal the hole walls of SiO2 were covered densely with large Ag particles whose surface was composed of lots of tiny Ag NPs. Especially at the top of the hole walls, six adjacent large Ag particles surrounded each hole. FDTD simulation demonstrated both the gaps between the tiny Ag NPs and those between large Ag particles were efficient hot spots, which could concentrate the electric field effectively. By using crystal violet (CV) as the probe molecule, the proposed Ag/SiO2 substrate could detect 10−9 M CV easily and the enhancement factor (EF) was calculated to be 1.7 × 107 due to the combined effect of hot spots and the 3D structure. The hexagonally arrayed nanohole structure endowed the proposed Ag/SiO2 substrate with good uniformity and the relative standard deviation (RSD) was estimated to be as low as 5.79%. The great potential of this substrate in practical applications was demonstrated by the successful detection of tricyclazole down to 0.01 ppm which lived up to the EU standard.