We report the observation of appreciable enhancement of fluorescence induced by large Au colloids on a solid support. Au colloids 40, 59, and 81 nm in radius were homogeneously deposited on glass substrates with varying average particle distances and interparticle coupling. The fluorescence enhancement was examined by using self-assembled monolayers of two different fluorophore - protein conjugates: fluorescein isothiocyanate - human serum albumin (FITC-HSA) and epicocconone (Deep Purple) - bovine serum albumin (DP-BSA). We demonstrated strong variations of fluorescence enhancement with both interparticle distance and particle size. The highest observed fluorescence enhancement for DP-BSA was 15 times with the 162 nm Au colloid and an average interparticle distance of 67 nm, estimated from the particle density in the SEM images. We analyzed the origin of such large enhancement observed for optimized nanoparticle size and distance. To this aim, we separated the effect of the increased excitation rate from the electromagnetic field locally enhanced by the interaction of incident light with the nanoparticles from the effect of higher quantum yield due to an increase of the intrinsic decay rate of the fluorophore. We also demonstrated that the Au colloid monolayers on glass surfaces have outstanding macroscopic homogeneity. This important feature will pave the way for the application of our substrates for ultrasensitive bioassays and similar applications in biotechnology and life sciences.