Arrays of sub-wavelength holes (nanoholes) in gold films were used as a substrate for enhanced fluorescence spectroscopy. Seven arrays of nanoholes with distinct periodicities (distances between the holes) were fabricated. The arrays were then spin-coated with polystyrene films containing different concentrations of the fluorescent dye oxazine 720. The dye was excited via resonant extraordinary transmission of the laser source through the nanoholes. Enhanced fluorescence was observed when the geometric characteristics of the arrays allowed for an enhancement in the transmitted excitation. This enhancement occurred via surface plasmon excitation by the laser and a consequential increase in the local electromagnetic field in a sub-wavelength region at the metal-film interface. It was demonstrated that the sensitivity of the fluorescence measurement (change in signal vs change in dye concentration in the polymer film) is significantly larger at the surface plasmon resonance conditions than that obtained from equivalent films on glass substrates. Enhancement factors for the fluorescence emission were calculated for each array, with a maximum enhancement of close to 2 orders of magnitude as compared to the emission of films on glass. The results presented here indicate that arrays of nanoholes are interesting substrates for the development of fluorescence sensors based on surface plasmon resonance, as they provide a platform that allows both spatial confinement and enhancement of excitation light. Moreover, the collinear characteristics of the present optical setup, due to the resonant extraordinary transmission through the nanohole arrays, are more conducive to miniaturization and chip integration than more traditional experimental geometries.