The surface-enhanced fluorescence properties of nanoporous gold (NPG) films were investigated by controlling the distance between substrate and fluorescent molecules via silica depositing. Silica was coated on the surface of NPG by physical vapor deposition, and the distance between NPG surface and fluorescent molecules were determined by adjusting the thickness of the silica film. Both the nanopore size and the distance between substrate and fluorescent molecules affect the properties of surface-enhanced fluorescence. As nanoporous gold is of a nanometer porous structure, the surface plasmon of NPG can enhance the excitation field of a molecule by the enhancement of the local electric field near the molecule so as to enhance the excitation intensity and efficiency of the molecule. When there is an appropriate distance between the fluorescent molecule and the NPG surface, the surface plasmon can enhance the electromagnetic field around NPG through the resonance coupling with the photon, and achieve the fluorescence enhancement. It is found that the fluorescence from the assembled Rhodamine 6G molecules can achieve the best enhancement with 20 nm silica coated NPG films, and the optimal ligament size of NPG is 36 nm.