We report on the enhancement of sensitivity of SPR biosensor by modifying the metal surface. A mixture layer, in which gold and dielectric medium coexist, was simply prepared by increasing the roughness of gold surface deposited onto a glass substrate via the reduction reaction. The absorbance spectrum and atomic force microscopy study showed that the average roughness of modified substrate increased by 4 times, compared to the unmodified substrate. The SPR curves of the unmodified substrate and the modified substrate were measured with the various refractive index of the dielectric medium. The sensitivity of modified substrate to the refractive index of dielectric medium was increased by 1.5 times, compared to the unmodified substrate. In result, the addition of the mixture layer between the gold surface and the dielectric medium by the reduction reaction can provides the enhancement of sensitivity in the SPR biosensor.