Ordered metal nanoparticle (NP) arrays have attracted tremendous attention for potential plasmonic applications due to their unique surface plasmon resonance. Herein, we fabricated highly ordered Ag NP arrays employing anodized aluminum (Al) templates and the vacuum thermal evaporation technique. The morphological evolution and corresponding optical property changes of the Ag NP arrays during annealing treatment has been investigated. It is found that there is a transition point approximately at annealing temperature of 290°C, where the shape of Ag NPs is most spherical and the peak position of quadrupole mode has a largest wavelength about 500 nm. Then, the surface-enhanced Raman spectra of Rhodamine 6G on Ag NP arrays annealed at different temperatures has been demonstrated at an excitation wavelength of 532 nm. The maximum SERS enhancement is also observed at 290°C. Our findings provide a way to further improve the performance of the plasmonic structures.