Quantitative analysis is of importance for surface-enhanced Raman scattering (SERS). However, due to fluctuations in the enhancing performance of the substrates, it is difficult to obtain reliable results. In this paper, a reliable quantitative method is introduced to overcome this problem with graphene on the top of Ag nanoholes structure as SERS substrates by an internal standard method. To achieve the internal standard method. Ag nanoholes are firstly prepared by surface plasmon (SP) lithography technology. Then a monolayer graphene is transferred onto the surface of the Ag nanoholes structure. 2D Raman peak of graphene is used as an internal standard to normalize the intensity of analyte molecules. The random representative and averaged Raman intensity of different concentration of rhodamine 6G (R6G) is collected with graphene/Ag nanoholes (GE/AgNHs) structures as SERS substrates, and the corresponding normalized intensity is also calculated and discussed in details. The relative standard deviation (RSD) is reduced from 25% (Raman intensity) to 12% (normalized intensity). The quantification of R6G is demonstrated down to the detection limit of 10(-15) M. (C) 2018 Optical Society of America under the terms of the OSA Open Access Publishing Agreement