Single-molecular surface-induced fluorescence attenuation (smSIFA) is a precise method of studying the vertical movement of biological macromolecules based on two-dimensional material receptors. This method is not affected by two-dimensional planar motion of membrane or proteins. However, the detection range and accuracy of vertical movement are determined by the properties of two-dimensional materials as receptors. In recent years, surface induced fluorescence attenuation based on graphene oxide and graphene has played an important role in studying biomacromolecules. However, the detection range of graphene and graphene oxide are limited owing to the fixed and limited characteristic quenching distance. Adjusting the detection range requires replacing the medium material, which poses difficulties in selecting and preparing materials. Therefore, it is urgently needed to develop controllable materials for single-molecular SIFA. In this study, the single-molecule SIFA with graphene oxide as the medium acceptor is improved by reducing graphene oxide through thermal reduction. By controlling the reduction temperature, reduced graphene oxides to different reduction degrees are prepared and the characteristic quenching distances are adjusted. The characteristic quenching distance is measured by fluorescent labeled DNA. Single-molecule SIFA based on reduced graphene oxide is used to observe the conformational changes of Holliday junction, and the detection range of reduced graphene oxide is demonstrated.