The surface enhanced fluorescence(SEF)detection bases by plasmonic nanopillars array with nanoparticles has opened up a new gate in the application of biological imaging and sensing. The fluorescence enhancement of the probe molecule depends on its position in equilibrium, which is close to the hot spot leading to the electromagnetic field enhancement, but not too close to the metal surface resulting in quenching. Here, a large scale SiO2-Ag-cicada wing SEF substrate was fabricated by magnetron sputtering with correction enhancement factor of 797.6. Thereinto the cicada wing provides the skeleton of the nanopillars array structure, the deposited Ag constructs two kinds of hot spots, and SiO2 forms a separation layer to prevent quenching. Moreover, the substrate exhibited good reproducibility, high sensitivity with low limits of detection (LOD) and high stability for oxidation resistance. We propose that SEF substrate with modification of SiO2 can not only improve the enhancement performance, but also expanding its application in the biological investigations.