This paper reports on the improved fabrication process and the optical characterization of different nano-hole arrays in thin metal films that are to be integrated into a novel atto-litre titre plate device for high-speed molecular analysis, such as DNA hybridizations and protein immunoassays. The optical detection is based on the enhanced optical transmission that was recently discovered when light passes through periodically distributed sub-wavelength apertures in optically thick metal films. The transmitted light has also small angular diffraction and well-defined spectral features. Using electron-beam (e-beam) lithography (EBL) and lift-off technique, various array structures with hole diameters ranging between 100 nm and 200 nm and different pitches were fabricated in a 200-nm thick layer of gold (Au), palladium (Pd), and gold/palladium (Au/Pd = 60/40) alloy on glass. Introducing Pd to Au, the grain size of the material is decreased, getting a more well-defined shape of the holes. The transmitted spectrum was measured through periodically and randomly distributed nano-holes in Au. Transmitted spectra were compared as well through similar subwavelength hole arrays in Au, Pd, and Au/Pd alloy. Moreover, the fluorescence of Rhodamine G6 (0.05 µM) was measured when using the transmitted light through periodical cavities in Au as the illumination source. It reveals a nine-fold increase in the fluorescent signal.