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The preparation of surface-enhanced fluorescence (SEF) substrates is often influenced by experimental strategies and factors such as the morphology and size of the nanostructures. In this study, using the natural reed leaves (R.L.) without any special pretreatment as the substrate, metal silver was modified by magnetron sputtering technology to prepare a stable and efficient SEF system. The abundant “hedgehog-like” protrusions on R.L. substrate surface can generate high-density “hot spots”, enhancement factor (EF) is up to 3345 times, and stability and reproducibility have been verified in many measurements. The contribution of the intervention of silver nanostructure to the radiation attenuation process of fluorescent molecules is analyzed with the aid of Jablonski diagrams. 3D finite difference time domain (FDTD) simulate the spatial electric field and “hot spots” distribution of the substrate, “hedgehog-like” protrusions structure generate multiple “hot spots”, which produce an excellent local surface plasmon resonance (LSPR) effect and provide higher fluorescence signal. Finally, R.L./Ag-35 substrate is used to detect crystal violet (CV), the detection limit is as low as 10-13 M, and this “hedgehog-like” SEF substrate provides a new strategy for the trace detection of CV has good practical application value.

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