For breaking through the sensitivity limitation of conventional surface plasmon resonance (SPR) biosensors, novel highly sensitive SPR biosensors with Au nanoparticles and nanogratings enhancement have been proposed recently. But in practice, these structures have obvious disadvantages. In this study, a nanohole based sensitivity enhancement SPR biosensor is proposed and the influence of different structural parameters on the performance is investigated by using rigorous coupled wave analysis (RCWA). Electromagnetic field distributions around the nanohole are also given out to directly explain the performance difference for various structural parameters. The results indicate that significant sensitivity increase is associated with localized surface plasmons (LSPs) excitation mediated by nanoholes. Except to outcome the weakness of other LSP based biosensors, larger resonance angle shift, reflectance amplitude, and sharper SPR curves’ width are obtained simultaneously under optimized structural parameters.