We investigated the simultaneous excitation of localized surface plasmons (LSPs) and propagating surface plasmons (PSPs) on a thin metallic film with an array of nanoholes for the enhancement of fluorescence intensity in heterogeneous bioassays. Experiments supported by simulations reveal that the co-excitation of PSP and LSP modes on the nanohole array in a Kretschmann configuration allows for fluorescence enhancement of about 10(2) as compared to a flat Au surface irradiated off-resonance. Moreover, this fluorescence signal was about 3-fold higher on the substrate supporting both PSPs and LSPs than that on a flat surface where only PSPs were resonantly excited. Simulations also indicated the highly directional fluorescence emission as well as the high fluorescence collection efficiency on the nanohole array substrate. Our contribution attempts to de-convolute the origin of this enhancement and identify further ways to maximize the efficiency of surface plasmon-enhanced fluorescence spectroscopy for implementation in ultra-sensitive bioassays.