Plasmonic nanohole arrays for biosensing applications have attracted tremendous attention because of their flexibility in optical signature design, high multiplexing capabilities, simple optical alignment setup, and high sensitivity. The quality of the metal film, including metal crystallinity and surface roughness, plays an important role in determining the sensing performance because the interaction between free electrons in the metal and incident light is strongly influenced by the metal surface morphology. We systematically investigated the influence of metal crystallinity-related morphologies on the sensing performance of plasmonic nanohole arrays after different metal deposition processes. We utilised several non-destructive nanoscale surface characterisation techniques to perform a quantitative and comparative analysis of the Au quality of the fabricated sensor. We found empirically how the surface roughness and grain sizes influence the permittivity of the Au film and thus the sensitivity of the fabricated sensor. Finally we confirmed that the deposition conditions that provide both low surface roughness and large metal grain sizes improve the sensitivity of the plasmonic sensor.