The ability to precisely control the pattern of metallic structures at the micro- and nanoscale for surface plasmon coupling has been demonstrated to be essential for signal enhancement in fields such as fluorescence and surface-enhanced Raman scattering. In the present study, a series of silver coated gratings with tailored duty ratio and depth and a periodical pitch of 400 nm are designed and implemented. The influence of the grating profile on plasmonic properties and the corresponding enhancement factor are investigated by angular scanning measurement of reflectivity and fluorescence intensity and by finite difference time domain simulation. The application of the substrate in the enhanced fluorescence imaging detection of labeled protein is also investigated. This substrate has a wide range of potential applications in areas including biodiagnostics, imaging, sensing, and photovoltaic cells.