In this paper, we developed a metal-enhanced fluorescence (MEF) substrate by modification of the commercially available surface enhanced Raman spectroscopy (SERS) substrate that may meet the reproducibility and sensitivity challenge of MEF. In spite of many studies and interest on MEF from a number of research groups, application to real-world situations and its commercial use remain challenging mainly due to the difficulties in fabricating reproducible MEF substrates. Specifically, one of the challenges is achieving a standardized MEF substrate for reproducible fluorescence intensity enhancement and/or changes in lifetime. The gold standard klarite substrates for SERS were coated with a thin layer of silver nanoparticles for MEF studies. To test the newly developed MEF substrates, a monolayer of streptavidin conjugated Alexa-647 was assembled on biotinylated-glass or MEF substrates. We observed over 50-fold increase in the fluorescence intensity from a monolayer of streptavidin conjugated Alexa-647 on the biotinylated MEF substrate compared to the same on glass substrate. A significant reduction in the lifetime and increased photostability of Alexa-647 on MEF substrate was observed. Fluorescence lifetime imaging was performed on the monolayer of dye assembled on the modified SERS substrates. We expect this study will serve as a platform to encourage the future use of a standardized MEF substrate for a plethora of sensing applications.