Surface plasmons at a metal/dielectric interface resonate with incident light, generating an evanescent field at the interface, which is highly sensitive to the change in refractive index of the medium. These characteristics are utilized as the basis for surface plasmon resonance-based sensors with Kretschmann configuration, providing label-free and real-time monitoring of binding interaction between probe and target moieties. Although graphene is recently extensively investigated in the field of optical sensors for the improvement of sensing performance, the proposed enhancement mechanisms in each study are ambiguous and inconsistent. Here, graphene-deposited Au film as advanced plasmonic sensing substrates is reported. Work function measurements of Au–graphene with different number of layer and doping state explicitly corroborate the mechanism of sensitivity increase, confirming that the enhanced refractive index sensitivity originates from induced surface dipole due to the charge transfer between Au film and graphene. The best sensitivity is attained from two layers of graphene by a chemical vapor deposition method. Biotinylated bovine serum albumin and streptavidin are used to evaluate the biosensing performance. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim