Surface plasmon resonance (SPR) based sensors play an important role in the biological and medical fields, and improving the sensitivity is a goal that has always been pursued. In this paper, a sensitivity enhancement scheme jointly employing MoS2nanoflower (MNF) and nanodiamond (ND) to co-engineer the plasmonic surface was proposed and demonstrated. The scheme could be easily implemented via physically depositing MNF and ND overlayers on the gold surface of an SPR chip, and the overlayer could be flexibly adjusted by controlling the deposition times, thus approaching the optimal performance. The bulk RI sensitivity was enhanced from 9682 to 12,219 nm/RIU under the optimal condition that successively deposited MNF and ND 1 and 2 times. The proposed scheme was proved in an IgG immunoassay, where the sensitivity was twice enhanced compared to the traditional bare gold surface. Characterization and simulation results revealed that the improvement arose from the enhanced sensing field and increased antibody loading via the deposited MNF and ND overlayer. At the same time, the versatile surface property of NDs allowed a specifically-functionalized sensor using the standard method compatible with a gold surface. Besides, the application for pseudorabies virus detection in serum solution was also demonstrated.