The crystallization of Au/glass ultrathin films for surface plasmon resonance (SPR) biosensor has been studied using synchrotron X-ray scattering and field emission scanning electron microscope. In films thinner than 30 nm, crystallized Au grains with  preferred orientation were formed in the as-deposited amorphous precursor. In film with 58-nm thickness, however, Au (200) powder grains existed on top of the Au (111)-oriented grains near the interface. At the annealing temperature of 400 degrees C, the Au (200) powder grains disappeared, while the Au (111)-oriented grains grew further. The behavior of the surface morphology of Au thin films post-annealed at 400 degrees C was consistent with the thickness dependence of the crystallization. In a 10-nm-thick film, the Au (111)-oriented grains fully crystallized, and then became separated with each other. By increasing to 30-nm film thickness, the Au (111)-oriented grains grew further, coalesced into large columnar-type grains, and showed smooth surface. We suggest that the appropriate thickness of Au/glass thin film for SPR biosensor need over 30-nm-thick, considering smooth surface.