Two-dimensional (2D) metal nanoparticle platforms hold great potential in high-throughput point-of-care testing on basis of their excellent fluorescence enhancement effect. Self-assembly of metal nanoparticles (NPs) is versatile but simple for the preparation of 2D platforms; however, the surface charges on NPs would repel each other and induce large gaps on platforms, which hinder the generation of intense hotspots in electromagnetic fields and thus weaken the fluorescence enhancement effect. Here, we presented the hierarchical assembly of large-sized silver (Ag) NPs and small-sized gold (Au) NPs for constructing fluorescence enhanced platforms. The small Au NPs with weak fluorescence were chosen to complementarily fill into the larger gaps of the pre-assembled Ag platforms for increasing the electromagnetic hotspots. The resulting Ag/Au hybrid platforms (1-, 3- and 6-Ag/Au) exhibited growing fluorescence enhancement effects and the maximum enhanced factor reached 3.6-fold. The inserting of small Au NPs enhanced the fluorescence emission of fluorescent dye (cyanine-5) on the Ag platforms, especially on the monolayer 1-Ag platform, which increased by 161.6%. Moreover, the finite-difference time-domain (FDTD) calculations revealed the underlying cause of the improved electromagnetic fields in the Ag/Au hierarchical architecture in comparison to the Ag or Au NP. This work presents the hierarchical assembly is expected to be a powerful tool in the large-scale fabrication of fluorescence enhanced detection platforms.