Great progress has been made in fluorescence-based detection utilizing solid state enhanced substrates in recent years. However, it is still difficult to achieve reliable substrates with tunable enhancement factors. The present work shows liquid fluorescence enhanced substrates consisting of suspensions of Fe3O4 nanoparticles (NPs), which can assemble 3D photonic crystal under the external magnetic field. The photonic bandgap induced by the equilibrium of attractive magnetic force and repulsive electrostatic force between adjacent Fe3O4 NPs is utilized to enhance fluorescence intensity of dye molecules (including R6G, RB, Cy5, DMTPS-DCV) in a reversible and controllable manner. The results show that a maximum of 12.3-fold fluorescence enhancement is realized in the 3D Fe3O4 NP substrates without the utilization of metal particles for PCs/DMTPS-DCV (1.0 × 10(-7) M, water fraction (f w) = 90%).