A two-dimensional (2D) plasmonic coupling nanostructure for enhanced fluorescence observation using a microscope is presented. The substrate contained periodically assembled nanohole arrays with a pitch of 400 nm and a depth of 25 nm. In comparison with one-dimensional (1D) gratings, this new substrate presented an excellent surface plasmon coupling ability to illuminate light from all directions. Under an optical microscope, an enhancement in the fluorescence intensity of up to 100 times compared with a plain glass slide was observed. The ability to markedly increase the fluorescence intensity means this technique has great potential for application in biodiagnostics, imaging, sensing, and photovoltaic cells.